CopyrightC) 1977 American Society for Microbiology Printed in U.S.A.
Selective and
Enhanced Recovery of Group
A
and
B
Streptococci from Throat Cultures with Sheep Blood Agar
Containing Sulfamethoxazole and Trimethoprim
BRUCE A. GUNN,* DAVID K. OHASHI, CHARLOTTE A. GAYDOS, AND EDITH S. HOLT Walter Reed Army Medical Center, Washington, D.C. 20012
Received forpublication 10 January 1977
Sheep blood agar containing 23.75 ,ug of sulfamethoxazole and 1.25 ,g of
trimethoprim (SXT-BA) per ml was compared with conventional sheep blood
agar (SBA) for isolatinggroup AandBstreptococcifrom throat cultures.This
selective mediumallowed much better recovery of group A and B streptococci
andsuppressed the growth of the normal flora, including "viridans" streptococci.
In aninitial study of 700 throat cultures, SXT-BA recovered 42% more group A
and49% moregroup Bstreptococci than did SBA. When SXT-BAwasintroduced
into the routine microbiology laboratory and used by a number of medical
technologists, SXT-BA recovered 28% more group A and 37% more group B
streptococcithan did SBA. Inaddition, the selective medium inhibited83% of
the non-group AandB streptococcithatwererecoveredby SBA.
The search for a more sensitive medium for
recoveryofgroup A beta-hemolytic streptococci
has continued (4, 6, 7, 12-14, 16). In the United
States, sheep bloodagar(SBA) is recommended
and used by mostclinical laboratories for
pri-mary isolation of these microorganisms. The
need fora more sensitive medium forrecovery
ofgroupAstreptococciisessential because
clin-icalassessment andtreatmentofa sorethroat
remains so uncertain. Recent studies have
shownthat selective media incorporating
var-ious inhibitors will insure better recovery of
these organisms.
Recovery ofgroup A streptococcifrom agars
and broth media hasimproved through the
in-corporation of agents such as sodium azide,
crystal violet, and antibiotics. These agents
haveveryeffectively eliminated the growth of
"diphtheroids," neisseria, staphylococci, and
gram-negative rods, but they generally have
notinhibited the growth of the "viridans"
strep-tococci. Nakamizo and Sato in 1972 (13)
suc-ceededininhibiting the "viridans" streptococci
and other members of the normal florawithan
ingenious mixtureof chemical agents.They
re-portedthat directplatingonSBAmissed 5%of
the positive cultures that were recovered by
direct plating on their selective agar. Vincent
etal. (16)incorporated neomycinandnalidixic
acid into SBA and showed that 3% of 66
beta-hemolyticgroup A streptococci weremissed by
SBA. With acommercially available medium,
Freeburgand Buckingham (7)foundthat
neo-mycin, nalidixic acid, and amphotericin B
im-provedrecovery ofgroup A streptococciby
in-hibiting certain members of the normal flora
that occasionally overgrew and obscured the
presence of beta-hemolytic streptococcal
colo-nies. Murray et al. (12), using gentamicin blood
agar, detected fewer group A but more
non-group A strains on their selective medium than
onSBA. These studies have clearly shown that
direct platingonselective agarmedia will
im-prove recovery of group A streptococci by no
more than 5% overthe number of isolates
de-tected by direct platingon SBA alone. Use of
multiple swabs may also help detect more
group Astreptococci (8 to 12%)thanwill direct
platingonSBA (10).
A recent review pointedoutthat several
in-vestigators, utilizing a variety of broth
enrich-mentmethods, demonstrated that 15 to 18%of
group A streptococci will not be recovered by
direct plating on SBA (6). Direct plating on
selective agar media will miss approximately
10%of the beta-hemolytic streptococci that the
selective broth methods will detect. Thus, the
bacteriologist should doeverything possible to
maximize isolation of group A streptococci
through proper use of selective agars and
broths.
Thisstudy was done to evaluate aselective
medium, SBA incorporating sulfamethoxazole
and trimethoprim (SXT-BA), for the isolation
of group A and B streptococci. Preliminary
studiesin ourlaboratory demonstratedthe
use-fulness of trimethoprim and sulfamethoxazole
to selectively reduce the normal flora while
allowing growthof group A and Bstreptococci.
MATERIALS AND METHODS
Conventional SBAwas prepared by adding 5 to 650
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7%defibrinated sheep blood to Trypticase soy agar (Difco). SXT-BA was made by the addition of 1.0 ml of a 25-mg/mlstock antibiotic solution to 1.0 liter of conventional SBA. The stock antibiotic solution was prepared by combining equal parts of solution A with solution B. Preparation of solution A involved dissolving 0.125 g of trimethoprim (Burroughs Well-come Co.) in a few milliliters of 0.1 NHCl and then bringing the volume up to 50 ml with distilled water. Solution B was prepared by dissolving 2.375 g of
sulfamethoxazole (Burroughs Wellcome Co.) in a
fewmilliliters of 1.0 N NaOH and thenbringingthe volume up to 50ml withdistilled water. The stock antibiotic solution was stored in 6.0-ml portions at
-30°Cand was not sterilized prior to use. The final
combined concentration oftrimethoprimand sulfa-methoxazole in SXT-BA was 25 ,ug/ml.
Each batch of SXT-BA was quality assured by testingforgrowthof the control organisms, Strepto-coccuspneumoniae (WRAMC06,Walter Reed Army Medical Center), S. pyogenes (ATCC 19615; BAC-TROL, Difco), and Proteus vulgaris (ATCC 13315; BACTROL, Difco). Streptococcus pyogenes grew as
beta-hemolyticcolonies on thismedium and served
as the positive control for both growth and beta-hemolysis. SXT-BA was prepared weekly and stored
at 5to 10°Cinplastic bagsuntil use.
Seven hundred throat cultures were obtained from both in-patients and out-patients at Walter Reed Army Medical Center, Washington, D.C. These cultures were accessioned as they were
re-ceivedfrom all the clinics and wards that submitted specimensduringthisstudy. Approximately40%of thecultures were obtained from pediatric patients. After these initial 700 cultures were tested to deter-minethe usefulness of SXT-BA to improve recovery of group A and B streptococci, an additional 5,500 throatcultures were inoculated to both mediaina
similar manner over a period of 5 months. This portionof thestudywasinitiated to determine the
abilityof themedium to be usedeffectively bybench
technologistsin amicrobiologylaboratory.
Throatcultures were taken with a sterile
rayon-tipped swab and transported ina modified Stuart
transportmedium (Culturette, Marion Health and
Safety, Inc.). The specimens were transported at
room temperature and processed within 16 h from thecollection time.
Throat culture swabs were inoculated onto SBA and thenSXT-BA. Inoculatedareas were streaked
forisolation,and the agar wasstabbed several times
forobservationof subsurfacehemolysis. The
inocu-latedplateswere incubated for 18 to 24 h at 35°C in
anatmosphereof5to 9%carbondioxide in air with a
relativehumidityof 40 to60%.Afterincubation,all plates were examined for the presence of beta-hemo-lytic colonies.
Allbeta-hemolytic streptococci were serologically grouped bycounterimmunoelectrophoresis and
pre-sumptivelygrouped by the bacitracin-SXT disk
sus-ceptibility test described elsewhere (8). To
deter-mine whether the antibiotics in the selective
me-diumwouldalter theoutcomeofthe bacitracin-SXT
susceptibilityresults,64group A andBstrains were
tested forsusceptibilitytobacitracin and SXT disks
onboth media. Zones of inhibitionweremeasuredto
the nearestmillimeter after 18 to24hof incubation
at350C.
RESULTS
Seven hundred throat cultures were plated
onbothSXT-BA and SBAduringaninitial
8-week periodofstudy.A total of 165 (24%)
cul-tures contained beta-hemolytic streptococci
when both media were used for isolation. Of
these, 55 were serotyped as group A, 33 as
groupB, and 77asgroups other than AorB.
Sixty-five percent of the group A streptococci
wereisolated fromchildren, but only33%of the
group B and 20%o of the other groupswere
re-covered from pediatric patients.
The antibiotics in SXT-BA effectively
re-duced thenormalflora, bothqualitatively and
quantitatively, when compared with SBA. Sixty-three (9%) of the cultures had luxurious growth of normal flora on SBA but did not grow
onSXT-BA. In all othercultures, the density of
growth ofviridans streptococci, staphylococci,
neisseria, diphtheroids, and gram-negative
rods on SXT-BA was reduced by 25 to 75%.
Growthof yeast was notaffected by the
selec-tivemedium, and, on four occasions, these
mi-croorganisms were isolatedin purecultureon
SXT-BA, whereas they were not observed on
SBA. Colonies of group A and B streptococci
were smaller on SXT-BA than on SBA, and
hemolytic zones around the colonies were
pro-portionately smaller (Fig. 1).
SBAmissed44%(25/55)of the group A
strep-tococcithatwererecovered by both media
(Ta-ble 1). Of these twenty-four strains cultured
onlyonSXT-BA,54%grewasgreaterthan 100
colonies oneachplate (Fig. 2). Of the
remain-ingcultures,33%grew asless than 10colonies
and 13% grew between 10 and 100 colonies.
SXT-BA missedonly 2% (1/55)of the group A
streptococci recovered by both media. When
this group Astrain wasinitiallyisolated, only
four beta-hemolytic colonies grew on the
pri-maryplate. Of the30group Astrainsthatwere
isolated onboth media,97%had morethan 100
coloniesonthe selectiveplate, but only70%of
the group A strains isolated on SBA grew as
luxuriantly. Overall, SXT-BA recovered 42%
moregroupAstreptococci than did SBA.These
findings were significant by chi square (P <
0.025).
SBAmissed61% (20/33) ofthe groupB
strep-tococci that were recovered with both media
usedfor isolation (Table 1). Twenty percent of
the strains that were recovered only on
SXT-BAhad more than 100 colonies on each plate.
Of theremainingcultures, 30%grewwith less
than 10colonies and50%grewbetween 10and
100colonies. SXT-BA missed12% (4/33) of the
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652 GUNN ET AL.
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ISOLATION OF GROUP A AND B 653
group B streptococci recovered by both media.
In general, the colony counts on SXT-BAwere
always greaterthan on SBA. Overall, SXT-BA
recovered 49% more group B streptococci than
did SBA. These findings also were significant
by chi square (P < 0.05).
The antibiotics present in SXT-BA sup-pressed the growth of 90% (69/77) of the
non-group A and B streptococci, except for eight
Lancefield group C strains.
After the first 700 throat cultures were
stud-ied, SXT-BA was usedin ourlaboratory along
with SBA asaroutine medium and was
clini-callycompared with SBA for screening throats
forbeta-hemolytic streptococci. In this study,
508groupA, 210 group B, and 269 other
groupa-ble beta-hemolytic streptococci were isolated
from 5,500 throat cultures. Approximately one-half of the streptococcal cultures were
presump-tively groupedby the bacitracin-SXTtest,and
the remaining cultures were serologically
grouped. In this study, SXT-BA recovered28%
more group A and 37% more group B
beta-hemolytic streptococci thandid SBA. Only 17%
ofthe269non-groupAandBstreptococci grew
ontheselectivemedium. SevengroupAstrains
grew on SBA but not SXT-BA. These strains
wereall resistanttoSXT andgrew onSXT-BA
uponsubculture. The number of observable
col-onies on SXT-BA was generally greater than
the number of colonies on SBA (Table 2).
DISCUSSION
This study clearly demonstrates that
SXT-BA improves the isolation of group A and B
streptococci significantly. This medium
in-hibits the growth of most normal flora,
includ-ingthe viridansstreptococci, tosuchan extent
thatbacterial competition is greatly modified
and recoveryof S. pyogenes is increased.
Comparedwith SXT-BA, SBA missed a
sig-nificant number of group A (P < 0.025) and
group B (P <0.05) streptococci. Although it is
welldocumentedinthe literature that selective
media will improve the recovery of groupAand
B streptococci (1, 2, 4, 6, 12, 13, 16), selective
agars arelessefficient than selective broths for
thispurpose. However, inthis study, SXT-BA
attained recoveryratescomparabletothe
selec-tivebroth enrichment media.
Beta-hemolytic colonies growing on SXT-BA
and their zones of hemolysis were always
smaller than whengrown upon SBA (Fig. 1).
TABLE 1. Number ofbeta-hemolytic streptococci recoveredfrom SBA and SXT-BA media
Group SBA SXT-BA
Both
me- Totalonly only dia
A 1 24 30 55
B 4 20 9 33
Othera 69 1 7 77
a Other, Beta-hemolytic streptococci other than
groupsA and B.
TABLE 2. Comparison of the number of beta-hemolytic groupAstreptococcal colonies recoveredon
SBA andSXT-BA Total No. of colonies Medium
cul-tures 0 1-10 11-100 100+
SBA 508 149 47 148 164
29%a 9% 29% 32%
SXT-BA 508 7 56 216 229
1% 11% 43% 45%
aPercentageof total cultures.
Although hemolytic colonies could usually be
subcultured directly on SBA for performance
ofthebacitracin-SXTtest, occasionally
subcul-tures to SXT-BA were necessary to obtain a
valid bacitracin-SXTtest.Apparently, the
nor-mal flora was inadvertently picked and
over-grewthe beta-hemolytic streptococci on SBA.
This observation again demonstrates the
inhib-itory natureof normal flora uponS. pyogenes.
The ecological relationships of
"nonpatho-genic" bacteriatogroupAstreptococciareboth
interesting and complex. Sanders in 1969 (15)
tested 111 strains ofStaphylococcus
epidermi-dis, 109diphtheroids, 100nonpathogenic
neis-seria, and 124 viridans streptococci for
inhibi-tion of S. pyogenes. Only viridans streptococci
(47%)werecapableof bacterial interference.
In 1975, Bill and Washington (3) described
inhibition of groupAstreptococcibya common
throatinhabitant,S. salivarius. Jaggetal. (9)
recently reviewed the literature concerning
bacteriocins of gram-positive bacteria and
listed the viridans streptococci as very potent
inhibitors of beta-hemolytic streptococci.
In-cluded among the causes for inhibition were
viridins (bacteriocins ofalpha-hemolytic
strep-tococci), toxic metabolic products (e.g.,
hydro-gen peroxide), substrate depletion, creationof
unphysiological environments, andprevention
FIG. 1. GroupAbeta-hemolytic streptococci growing luxuriantlyon both isolation media. (A)SBA; (B) SXT-BA.
FIG. 2. Normalthroat flora growing on SBA (A). Same throat culture showing groupA beta-hemolytic streptococcigrowingonlyonSXT-BA (B).
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of beta-hemolysisexpression by peroxides (5, 9, 15, 16).
Many of the bacterial interactions that occur
in vivo also occur on plated media. Croweand
Sanders (5) reported that colonization of
chil-dren withgroup A streptococci varied according
tothe degree of bacterial interferencethat the
normal oropharyngeal flora of each child was
capable ofexerting. Under the selective
pres-sure of colonization inhibitory organisms
in-creased. He suggested that this selective
proc-essmaybe themechanism whereby adults
ulti-matelybecome more resistant than children to
streptococcal infection. From these studies it
would be assumed that the expression of
growth and beta-hemolysis of group A
strepto-cocci growing amongnonpathogenic
microorga-nisms would be influenced by the percentage of
the total number ofbacteria capable of exerting
bacterial interference. To maximize the
recov-ery of S. pyogenes from throat cultures, it
would benecessary toeliminateasmuch of the
inhibitory flora as possible. Since the viridans
streptococci have most often been associated
withthe inhibition ofgroup A streptococci,
se-lective media should be designed to suppress
their growth. This has notbeen thecase,
how-ever, primarily because those agents thatare
capable of inhibiting the viridans streptococci
usually inhibit S.pyogenes. Inthisstudy, SXT
antibioticswereshown tobeexceptions.
Kaplan(10) in 1972reported studiesinwhich
the degree of positivityofthroat cultures was
notentirely reliableforidentifying the acutely
infected individual and differentiating an
acutelyinfected patient fromanasymptomatic
carrier. Quite possibly the reason that the
de-greeof positivity of cultures hasnotcorrelated
with the degree of significant clinical illness
hasbeen dueto the failure of selective broths
and agars to adequately grow beta-hemolytic
coloniesas does SXT-BA.
Beta-hemolytic colonies onSXT-BAwere
al-waysobservedintheprimaryinoculationareas
duetothe suppression ofsomuch normal flora
(Fig. 2). In comparison, beta-hemolyticcolonies
wereonly occasionally isolatedinthe primary
inoculation areasof SBA. These SBA findings
have also been observedinourlaboratory with
selective agar media other than SXT-BA. It
becomes quiteevident when SXT-BA platesare
compared with SBA plates that the observable
number ofcoloniesonSBAdoesnotreflect the
actual numbers ofbeta-hemolytic streptococci
thatwere present onthe original swab (Fig. 2).
The selective plates more reliablypresent the
degree of throat involvement than do SBA
plates by reducing thenormal flora that tends
to overgrow, mask, and/or inhibit the
beta-hemolytic streptococci.
The one group A strain that grew only on
SBA presented four observable beta-hemolytic
colonies on the primary plate. Whatwas
sur-prising about this isolatewas that there were
absolutelynobeta-hemolytic colonies on
SXT-BA. In other cultures, coloniesonthe selective
mediumwere always equalto andin most
in-stancesgreaterthan the number of colonieson
SBA. Inoculation of the SBAplatesbeforethe
selective plates could possibly explain the
re-coveryof thisstrainonlyonSBA, especially in
view ofthe finding of others (10) that use of
singleswabs is far less than optimum for
maxi-mumrecoveryofgroup Astreptococci.
An additional advantage of SXT-BA over
other selectivemedia isthe inhibition of
beta-hemolyticstreptococciother thangroups Aand
B. Although non-group A streptococci have
been isolated from patients with pharyngitis, only rarely have they been associated with
rheumatic fever or acute glomerulonephritis
(11). By not recoveryingthesemicroorganisms,
SXT-BAaccomplishestwo objectives. First, by
reducing the number of bacitracin tests to be
performed, SXT-BAsaves both technical time
and laboratory media. Second, by inhibiting
manyof the bacitracin-susceptiblenon-group A
streptococci, the treatment of non-group A
pharyngitisisreduced, and, therefore, the
pos-sibility of allergic reactions to penicillin is
somewhat lessened. Thatgroup B streptococci
are isolated on SXT-BA along with group A
strains is not considered a disadvantage, as
preliminary studies in our laboratory indicate
that SXT-BAmaybeusedas ageneralpurpose
medium for the isolation of group A and B
streptococci from a variety of clinical
speci-mens.
The final study of5,500throat cultures
dem-onstrated that SXT-BAcanbeusedby
technol-ogists possessing varyingbacteriologicalskills.
Duringthis portion of the study it was
deter-mined thatbeta-hemolytic coloniesonSXT-BA
primaryplates should always be subculturedto
fresh SXT-BA plates for performance of the
bacitracin-SXTtest. The normal florasooften
overgrew the beta-hemolytic colonies when
subcultured to SBA that the bacitracin-SXT
testhadtoberepeatedon numerousoccasions.
It has shown during thisstudy that SXT disk
results on the antibiotic-impregnated agar
were notalteredbyincreased concentrations of
trimethoprim and sulfamethoxazole when
com-paredwith resultsobtainedonSBA. Inhibition
zonesaround bacitracin disksonSXT-BAwere,
ontheaverage, 4mmgreater in diameter than
thosezones onSBA.
Although the recovery of group A and B
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ISOLATION OF GROUP A AND B STREPTOCOCCI 655
streptococciduring the trial study varied
some-what from the initial study of 700 throat
cul-tures, SXT-BA still maintained its superiority
over SBA and was comparable to selective
broth techniques for therecovery of these
strep-tococci. It was not only shown that SXT-BA
improved the recovery ofgroup A and B
strep-tococci, butthe selective medium also increased
the quantity of colonies observed over those
observed on SBA. The seven strains of group A
streptococciisolated only on SBA presented less
than 10 observable colonies on the non-selective medium. As mentioned previously, these
cul-turescould be accounted for by the inoculation
method as well as by the use of single rather
thanduplicate swabs. All seven of these strains
were resistant to SXT by the bacitracin-SXT
test and were able to grow on SXT-BA upon
subculture.
From the results of this study, SXT-BA is
superior to directplatingonSBAforisolating
group A and B streptococci from throat
cul-tures.The mediuminhibits,tovarying extents,
all species of normal
oropharyngeal
flora aswell as mostbeta-hemolytic streptococci not of
groups A and B. Complete relianceon SBAas
the sole means of recoverying group A
beta-hemolytic streptococci isfar less than optimum,
and the use of selective media, such as
SXT-BA, to maximize recovery of these pathogens
should be utilizedinthe clinicallaboratory.
ACKNOWLEDGMENTS
Theable assistance of Wilbert Middleton and Massie Johnsoninthepreparation of the SXT-BA mediumis grate-fullyacknowledged. The helpfulcommentsofJ. M. Hard-manandN.I. Germanduring the preparation ofthis manu-scriptareappreciated.
LITERATURE CITED
1. Baker, C. J., D. J. Clark, and F. F. Barrett. 1973. Selective broth medium for isolation ofgroup B strep-tococci.Appl.Microbiol. 26:884-885.
2. Baker, C. J., D. K.Goroff, S.L.Alpert,C.Hayes,and W. M.McCormack.1976.Comparison of
bacteriologi-calmethods for the isolation of group B Streptococcus from vaginal cultures. J. Clin. Microbiol. 4:46-48. 3. Bill, N. J., and J. A. Washington. 1975. Bacterial
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4. Black, W. A., and F. Van Buskirk. 1973. Gentamicin blood agar used as a general-purpose selective me-dium.Appl.Microbiol. 25:905-907.
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6. Facklam,R. R. 1976. Areviewofthemicrobiological techniques for the isolation and identification of streptococci.Clin.Rev.Clin.Lab.Sci.6:287-317. 7. Freeburg, P. W., and J. M.Buckingham.1976.
Evalua-tionof the Bacti-Labstreptococciculturesystemsfor selectiverecoveryandidentificationof group A strep-tococci. J. Clin.Microbiol.3:443-448.
8. Gunn, B. A. 1976. SXT and Taxo Adisk for presumptive identification ofgroup Aand B streptococci in throat cultures.J. Clin.Microbiol.4:192-193.
9.Jagg, J. R., A.S. Dajani, and L. W. Wannamaker. 1976. Bacteriocins ofgram-positivebacteria. Bacte-riol. Rev. 40:722-756.
10. Kaplan, E. L. 1972.Unresolved problemsindiagnosis andepidemiologyofstreptococcal infection,p. 567-570.In L. W. Wannamaker andJ. M. Matsen(ed.), Streptococci and streptococcal diseases. Academic Press Inc., New York.
11. Moody, M.D. 1972.Old andnewtechniquesforrapid identificationof group A streptococci, p. 177-188. In L. W.WannamakerandJ. M. Matsen(ed.), Strepto-cocciandstreptococcal diseases. Academic PressInc., New York.
12. Murray, P. R., W. D.Wold,C. A.Schreck,and J. A. Washington. 1976.Effectsof selectivemedia and at-mosphere of incubationon the isolation of group A streptococci. J.Clin. Microbiol.4:54-56.
13. Nakamizo, Y., and M. Sato. 1972. New selective me-diumfor theisolation ofStreptococcushemolyticus. Am. J. Clin. Pathol.57:228-235.
14. Rosner, R. 1966. A new in vitro gram negative rod inhibitingagent which does notinterfere withthe growthofstreptococci. Am. J. Med. Technol. 32:69-73.
15. Sanders,E. 1969. Bacterial interference. I. Its occur-rence amongtherespiratory tract floraand charac-terization of inhibition of group A streptococci by viri-dans streptococci.J.Infect.Dis.120:698-707. 16. Vincent,W.F.,W. E.Gibbons, and H. A. Gaafar.1971.
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