JOURNAL OF CLINICAL MICROBIOLOGY, June 1977, p. 650-655Copyright C) 1977 American Society for Microbiology

Vol. 5, No. 6Printed in U.S.A.

Selective and Enhanced Recovery of Group A and BStreptococci from Throat Cultures with Sheep Blood Agar

Containing Sulfamethoxazole and TrimethoprimBRUCE A. GUNN,* DAVID K. OHASHI, CHARLOTTE A. GAYDOS, AND EDITH S. HOLT

Walter Reed Army Medical Center, Washington, D.C. 20012

Received for publication 10 January 1977

Sheep blood agar containing 23.75 ,ug of sulfamethoxazole and 1.25 ,g oftrimethoprim (SXT-BA) per ml was compared with conventional sheep bloodagar (SBA) for isolating group A and B streptococci from throat cultures. Thisselective medium allowed much better recovery of group A and B streptococciand suppressed the growth of the normal flora, including "viridans" streptococci.In an initial study of 700 throat cultures, SXT-BA recovered 42% more group Aand 49% more group B streptococci than did SBA. When SXT-BA was introducedinto the routine microbiology laboratory and used by a number of medicaltechnologists, SXT-BA recovered 28% more group A and 37% more group Bstreptococci than did SBA. In addition, the selective medium inhibited 83% ofthe non-group A and B streptococci that were recovered by SBA.

The search for a more sensitive medium forrecovery of group A beta-hemolytic streptococcihas continued (4, 6, 7, 12-14, 16). In the UnitedStates, sheep blood agar (SBA) is recommendedand used by most clinical laboratories for pri-mary isolation of these microorganisms. Theneed for a more sensitive medium for recoveryof group A streptococci is essential because clin-ical assessment and treatment of a sore throatremains so uncertain. Recent studies haveshown that selective media incorporating var-ious inhibitors will insure better recovery ofthese organisms.Recovery of group A streptococci from agars

and broth media has improved through the in-corporation of agents such as sodium azide,crystal violet, and antibiotics. These agentshave very effectively eliminated the growth of"diphtheroids," neisseria, staphylococci, andgram-negative rods, but they generally havenot inhibited the growth ofthe "viridans" strep-tococci. Nakamizo and Sato in 1972 (13) suc-ceeded in inhibiting the "viridans" streptococciand other members of the normal flora with aningenious mixture of chemical agents. They re-ported that direct plating on SBA missed 5% ofthe positive cultures that were recovered bydirect plating on their selective agar. Vincentet al. (16) incorporated neomycin and nalidixicacid into SBA and showed that 3% of 66 beta-hemolytic group A streptococci were missed bySBA. With a commercially available medium,Freeburg and Buckingham (7) found that neo-mycin, nalidixic acid, and amphotericin B im-proved recovery of group A streptococci by in-hibiting certain members of the normal flora

that occasionally overgrew and obscured thepresence of beta-hemolytic streptococcal colo-nies. Murray et al. (12), using gentamicin bloodagar, detected fewer group A but more non-group A strains on their selective medium thanon SBA. These studies have clearly shown thatdirect plating on selective agar media will im-prove recovery of group A streptococci by nomore than 5% over the number of isolates de-tected by direct plating on SBA alone. Use ofmultiple swabs may also help detect moregroup A streptococci (8 to 12%) than will directplating on SBA (10).A recent review pointed out that several in-

vestigators, utilizing a variety of broth enrich-ment methods, demonstrated that 15 to 18% ofgroup A streptococci will not be recovered bydirect plating on SBA (6). Direct plating onselective agar media will miss approximately10% of the beta-hemolytic streptococci that theselective broth methods will detect. Thus, thebacteriologist should do everything possible tomaximize isolation of group A streptococcithrough proper use of selective agars andbroths.

This study was done to evaluate a selectivemedium, SBA incorporating sulfamethoxazoleand trimethoprim (SXT-BA), for the isolationof group A and B streptococci. Preliminarystudies in our laboratory demonstrated the use-fulness of trimethoprim and sulfamethoxazoleto selectively reduce the normal flora whileallowing growth of group A and B streptococci.

MATERIALS AND METHODSConventional SBA was prepared by adding 5 to

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ISOLATION OF GROUP A AND B STREPTOCOCCI 651

7% defibrinated sheep blood to Trypticase soy agar(Difco). SXT-BA was made by the addition of 1.0 mlof a 25-mg/ml stock antibiotic solution to 1.0 liter ofconventional SBA. The stock antibiotic solution wasprepared by combining equal parts of solution Awith solution B. Preparation of solution A involveddissolving 0.125 g oftrimethoprim (Burroughs Well-come Co.) in a few milliliters of 0.1 N HCl and thenbringing the volume up to 50 ml with distilled water.Solution B was prepared by dissolving 2.375 g ofsulfamethoxazole (Burroughs Wellcome Co.) in afew milliliters of 1.0 N NaOH and then bringing thevolume up to 50 ml with distilled water. The stockantibiotic solution was stored in 6.0-ml portions at-30°C and was not sterilized prior to use. The finalcombined concentration of trimethoprim and sulfa-methoxazole in SXT-BA was 25 ,ug/ml.Each batch of SXT-BA was quality assured by

testing for growth of the control organisms, Strepto-coccus pneumoniae (WRAMC 06, Walter Reed ArmyMedical Center), S. pyogenes (ATCC 19615; BAC-TROL, Difco), and Proteus vulgaris (ATCC 13315;BACTROL, Difco). Streptococcus pyogenes grew asbeta-hemolytic colonies on this medium and servedas the positive control for both growth and beta-hemolysis. SXT-BA was prepared weekly and storedat 5 to 10°C in plastic bags until use.Seven hundred throat cultures were obtained

from both in-patients and out-patients at WalterReed Army Medical Center, Washington, D.C.These cultures were accessioned as they were re-ceived from all the clinics and wards that submittedspecimens during this study. Approximately 40% ofthe cultures were obtained from pediatric patients.After these initial 700 cultures were tested to deter-mine the usefulness of SXT-BA to improve recoveryof group A and B streptococci, an additional 5,500throat cultures were inoculated to both media in asimilar manner over a period of 5 months. Thisportion of the study was initiated to determine theability ofthe medium to be used effectively by benchtechnologists in a microbiology laboratory.

Throat cultures were taken with a sterile rayon-tipped swab and transported in a modified Stuarttransport medium (Culturette, Marion Health andSafety, Inc.). The specimens were transported atroom temperature and processed within 16 h fromthe collection time.

Throat culture swabs were inoculated onto SBAand then SXT-BA. Inoculated areas were streakedfor isolation, and the agar was stabbed several timesfor observation of subsurface hemolysis. The inocu-lated plates were incubated for 18 to 24 h at 35°C inan atmosphere of 5 to 9% carbon dioxide in air with arelative humidity of 40 to 60%. After incubation, allplates were examined for the presence ofbeta-hemo-lytic colonies.

All beta-hemolytic streptococci were serologicallygrouped by counterimmunoelectrophoresis and pre-sumptively grouped by the bacitracin-SXT disk sus-ceptibility test described elsewhere (8). To deter-mine whether the antibiotics in the selective me-dium would alter the outcome of the bacitracin-SXTsusceptibility results, 64 group A and B strains weretested for susceptibility to bacitracin and SXT diskson both media. Zones of inhibition were measured to

the nearest millimeter after 18 to 24 h of incubationat 350C.

RESULTS

Seven hundred throat cultures were platedon both SXT-BA and SBA during an initial 8-week period of study. A total of 165 (24%) cul-tures contained beta-hemolytic streptococciwhen both media were used for isolation. Ofthese, 55 were serotyped as group A, 33 asgroup B, and 77 as groups other than A or B.

Sixty-five percent of the group A streptococciwere isolated from children, but only 33% ofthegroup B and 20%o of the other groups were re-covered from pediatric patients.The antibiotics in SXT-BA effectively re-

duced the normal flora, both qualitatively andquantitatively, when compared with SBA.Sixty-three (9%) of the cultures had luxuriousgrowth ofnormal flora on SBA but did not growon SXT-BA. In all other cultures, the density ofgrowth of viridans streptococci, staphylococci,neisseria, diphtheroids, and gram-negativerods on SXT-BA was reduced by 25 to 75%.Growth of yeast was not affected by the selec-tive medium, and, on four occasions, these mi-croorganisms were isolated in pure culture onSXT-BA, whereas they were not observed onSBA. Colonies of group A and B streptococciwere smaller on SXT-BA than on SBA, andhemolytic zones around the colonies were pro-portionately smaller (Fig. 1).SBA missed 44% (25/55) of the group A strep-

tococci that were recovered by both media (Ta-ble 1). Of these twenty-four strains culturedonly on SXT-BA, 54% grew as greater than 100colonies on each plate (Fig. 2). Of the remain-ing cultures, 33% grew as less than 10 coloniesand 13% grew between 10 and 100 colonies.SXT-BA missed only 2% (1/55) of the group Astreptococci recovered by both media. Whenthis group A strain was initially isolated, onlyfour beta-hemolytic colonies grew on the pri-mary plate. Of the 30 group A strains that wereisolated on both media, 97% had more than 100colonies on the selective plate, but only 70% ofthe group A strains isolated on SBA grew asluxuriantly. Overall, SXT-BA recovered 42%more group A streptococci than did SBA. Thesefindings were significant by chi square (P <0.025).SBA missed 61% (20/33) of the group B strep-

tococci that were recovered with both mediaused for isolation (Table 1). Twenty percent ofthe strains that were recovered only on SXT-BA had more than 100 colonies on each plate.Of the remaining cultures, 30% grew with lessthan 10 colonies and 50% grew between 10 and100 colonies. SXT-BA missed 12% (4/33) of the

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group B streptococci recovered by both media.In general, the colony counts on SXT-BA werealways greater than on SBA. Overall, SXT-BArecovered 49% more group B streptococci thandid SBA. These findings also were significantby 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 eightLancefield group C strains.

After the first 700 throat cultures were stud-ied, SXT-BA was used in our laboratory alongwith SBA as a routine medium and was clini-cally compared with SBA for screening throatsfor beta-hemolytic streptococci. In this study,508 group A, 210 group B, and 269 other groupa-ble beta-hemolytic streptococci were isolatedfrom 5,500 throat cultures. Approximately one-halfofthe streptococcal cultures were presump-tively grouped by the bacitracin-SXT test, andthe remaining cultures were serologicallygrouped. In this study, SXT-BA recovered 28%more group A and 37% more group B beta-hemolytic streptococci than did SBA. Only 17%of the 269 non-group A and B streptococci grewon the selective medium. Seven group A strainsgrew on SBA but not SXT-BA. These strainswere all resistant to SXT and grew on SXT-BAupon subculture. The number of observable col-onies on SXT-BA was generally greater thanthe number of colonies on SBA (Table 2).

DISCUSSIONThis study clearly demonstrates that SXT-

BA improves the isolation of group A and Bstreptococci significantly. This medium in-hibits the growth of most normal flora, includ-ing the viridans streptococci, to such an extentthat bacterial competition is greatly modifiedand recovery of S. pyogenes is increased.Compared with SXT-BA, SBA missed a sig-

nificant number of group A (P < 0.025) andgroup B (P < 0.05) streptococci. Although it iswell documented in the literature that selectivemedia will improve the recovery ofgroup A andB streptococci (1, 2, 4, 6, 12, 13, 16), selectiveagars are less efficient than selective broths forthis purpose. However, in this study, SXT-BAattained recovery rates comparable to the selec-tive broth enrichment media.

Beta-hemolytic colonies growing on SXT-BAand their zones of hemolysis were alwayssmaller than when grown upon SBA (Fig. 1).

TABLE 1. Number of beta-hemolytic streptococcirecovered from SBA and SXT-BA media

Group SBA SXT-BA Both me- Totalonly only dia

A 1 24 30 55B 4 20 9 33

Othera 69 1 7 77a Other, Beta-hemolytic streptococci other than

groups A and B.

TABLE 2. Comparison of the number of beta-hemolytic groupA streptococcal colonies recovered on

SBA and SXT-BA

Total No. of coloniesMedium cul-

tures 0 1-10 11-100 100+

SBA 508 149 47 148 16429%a 9% 29% 32%

SXT-BA 508 7 56 216 2291% 11% 43% 45%

a Percentage of total cultures.

Although hemolytic colonies could usually besubcultured directly on SBA for performanceof the bacitracin-SXT test, occasionally subcul-tures to SXT-BA were necessary to obtain avalid bacitracin-SXT test. Apparently, the nor-mal flora was inadvertently picked and over-grew the beta-hemolytic streptococci on SBA.This observation again demonstrates the inhib-itory nature of normal flora upon S. pyogenes.The ecological relationships of "nonpatho-

genic" bacteria to group A streptococci are bothinteresting and complex. Sanders in 1969 (15)tested 111 strains of Staphylococcus epidermi-dis, 109 diphtheroids, 100 nonpathogenic neis-seria, and 124 viridans streptococci for inhibi-tion of S. pyogenes. Only viridans streptococci(47%) were capable of bacterial interference.

In 1975, Bill and Washington (3) describedinhibition of group A streptococci by a commonthroat inhabitant, S. salivarius. Jagg et al. (9)recently reviewed the literature concerningbacteriocins of gram-positive bacteria andlisted the viridans streptococci as very potentinhibitors of beta-hemolytic streptococci. In-cluded among the causes for inhibition wereviridins (bacteriocins of alpha-hemolytic strep-tococci), toxic metabolic products (e.g., hydro-gen peroxide), substrate depletion, creation ofunphysiological environments, and prevention

FIG. 1. Group A beta-hemolytic streptococci growing luxuriantly on both isolation media. (A) SBA; (B)SXT-BA.

FIG. 2. Normal throat flora growing on SBA (A). Same throat culture showing group A beta-hemolyticstreptococci growing only on SXT-BA (B).

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654 GUNN ET AL.

of beta-hemolysis expression by peroxides (5, 9,15, 16).Many of the bacterial interactions that occur

in vivo also occur on plated media. Crowe andSanders (5) reported that colonization of chil-dren with group A streptococci varied accordingto the degree of bacterial interference that thenormal oropharyngeal flora of each child wascapable of exerting. Under the selective pres-sure of colonization inhibitory organisms in-creased. He suggested that this selective proc-ess may be the mechanism whereby adults ulti-mately become more resistant than children tostreptococcal infection. From these studies itwould be assumed that the expression ofgrowth and beta-hemolysis of group A strepto-cocci growing among nonpathogenic microorga-nisms would be influenced by the percentage ofthe total number of bacteria capable of exertingbacterial interference. To maximize the recov-ery of S. pyogenes from throat cultures, itwould be necessary to eliminate as much of theinhibitory flora as possible. Since the viridansstreptococci have most often been associatedwith the inhibition of group A streptococci, se-lective media should be designed to suppresstheir growth. This has not been the case, how-ever, primarily because those agents that arecapable of inhibiting the viridans streptococciusually inhibit S. pyogenes. In this study, SXTantibiotics were shown to be exceptions.Kaplan (10) in 1972 reported studies in which

the degree of positivity of throat cultures wasnot entirely reliable for identifying the acutelyinfected individual and differentiating anacutely infected patient from an asymptomaticcarrier. Quite possibly the reason that the de-gree of positivity of cultures has not correlatedwith the degree of significant clinical illnesshas been due to the failure of selective brothsand agars to adequately grow beta-hemolyticcolonies as does SXT-BA.

Beta-hemolytic colonies on SXT-BA were al-ways observed in the primary inoculation areasdue to the suppression of so much normal flora(Fig. 2). In comparison, beta-hemolytic colonieswere only occasionally isolated in the primaryinoculation areas of SBA. These SBA findingshave also been observed in our laboratory withselective agar media other than SXT-BA. Itbecomes quite evident when SXT-BA plates arecompared with SBA plates that the observablenumber of colonies on SBA does not reflect theactual numbers of beta-hemolytic streptococcithat were present on the original swab (Fig. 2).The selective plates more reliably present thedegree of throat involvement than do SBAplates by reducing the normal flora that tendsto overgrow, mask, and/or inhibit the beta-

hemolytic streptococci.The one group A strain that grew only on

SBA presented four observable beta-hemolyticcolonies on the primary plate. What was sur-prising about this isolate was that there wereabsolutely no beta-hemolytic colonies on SXT-BA. In other cultures, colonies on the selectivemedium were always equal to and in most in-stances greater than the number of colonies onSBA. Inoculation of the SBA plates before theselective plates could possibly explain the re-covery of this strain only on SBA, especially inview of the finding of others (10) that use ofsingle swabs is far less than optimum for maxi-mum recovery of group A streptococci.An additional advantage of SXT-BA over

other selective media is the inhibition of beta-hemolytic streptococci other than groups A andB. Although non-group A streptococci havebeen isolated from patients with pharyngitis,only rarely have they been associated withrheumatic fever or acute glomerulonephritis(11). By not recoverying these microorganisms,SXT-BA accomplishes two objectives. First, byreducing the number of bacitracin tests to beperformed, SXT-BA saves both technical timeand laboratory media. Second, by inhibitingmany of the bacitracin-susceptible non-group Astreptococci, the treatment of non-group Apharyngitis is reduced, and, therefore, the pos-sibility of allergic reactions to penicillin issomewhat lessened. That group B streptococciare isolated on SXT-BA along with group Astrains is not considered a disadvantage, aspreliminary studies in our laboratory indicatethat SXT-BA may be used as a general purposemedium for the isolation of group A and Bstreptococci from a variety of clinical speci-mens.The final study of 5,500 throat cultures dem-

onstrated that SXT-BA can be used by technol-ogists possessing varying bacteriological skills.During this portion of the study it was deter-mined that beta-hemolytic colonies on SXT-BAprimary plates should always be subcultured tofresh SXT-BA plates for performance of thebacitracin-SXT test. The normal flora so oftenovergrew the beta-hemolytic colonies whensubcultured to SBA that the bacitracin-SXTtest had to be repeated on numerous occasions.It has shown during this study that SXT diskresults on the antibiotic-impregnated agarwere not altered by increased concentrations oftrimethoprim and sulfamethoxazole when com-pared with results obtained on SBA. Inhibitionzones around bacitracin disks on SXT-BA were,on the average, 4 mm greater in diameter thanthose zones on SBA.Although the recovery of group A and B

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ISOLATION OF GROUP A AND B STREPTOCOCCI 655

streptococci during the trial study varied some-what from the initial study of 700 throat cul-tures, SXT-BA still maintained its superiorityover SBA and was comparable to selectivebroth techniques for the recovery ofthese strep-tococci. It was not only shown that SXT-BAimproved the recovery of group A and B strep-tococci, but the selective medium also increasedthe quantity of colonies observed over thoseobserved on SBA. The seven strains of group Astreptococci isolated only on SBA presented lessthan 10 observable colonies on the non-selectivemedium. As mentioned previously, these cul-tures could be accounted for by the inoculationmethod as well as by the use of single ratherthan duplicate swabs. All seven ofthese strainswere resistant to SXT by the bacitracin-SXTtest and were able to grow on SXT-BA uponsubculture.From the results of this study, SXT-BA is

superior to direct plating on SBA for isolatinggroup A and B streptococci from throat cul-tures. The medium inhibits, to varying extents,all species of normal oropharyngeal flora aswell as most beta-hemolytic streptococci not ofgroups A and B. Complete reliance on SBA asthe sole means of recoverying group A beta-hemolytic streptococci is far less than optimum,and the use of selective media, such as SXT-BA, to maximize recovery of these pathogensshould be utilized in the clinical laboratory.

ACKNOWLEDGMENTSThe able assistance of Wilbert Middleton and Massie

Johnson in the preparation ofthe SXT-BA medium is grate-fully acknowledged. The helpful comments of J. M. Hard-man and N. I. German during the preparation ofthis manu-script are appreciated.

LITERATURE CITED1. 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, andW. M. McCormack. 1976. Comparison ofbacteriologi-

cal methods for the isolation of group B Streptococcusfrom vaginal cultures. J. Clin. Microbiol. 4:46-48.

3. Bill, N. J., and J. A. Washington. 1975. Bacterial inter-ference by Streptococcus salivarius. Am. J. Clin. Pa-thol. 64:116-120.

4. Black, W. A., and F. Van Buskirk. 1973. Gentamicinblood agar used as a general-purpose selective me-dium. Appl. Microbiol. 25:905-907.

5. Crowe, C. C., and W. E. Sanders. 1973. Bacterial inter-ference. II. Role of the normal throat flora in preven-tion of colonization by group A Streptococcus. J. In-fect. Dis. 128:527-532.

6. Facklam, R. R. 1976. A review of the microbiologicaltechniques for the isolation and identification ofstreptococci. Clin. Rev. Clin. Lab. Sci. 6:287-317.

7. Freeburg, P. W., and J. M. Buckingham. 1976. Evalua-tion of the Bacti-Lab streptococci culture systems forselective recovery and identification of group A strep-tococci. J. Clin. Microbiol. 3:443-448.

8. Gunn, B. A. 1976. SXT and Taxo A disk for presumptiveidentification of group A and B streptococci in throatcultures. J. Clin. Microbiol. 4:192-193.

9. Jagg, J. R., A. S. Dajani, and L. W. Wannamaker.1976. Bacteriocins of gram-positive bacteria. Bacte-riol. Rev. 40:722-756.

10. Kaplan, E. L. 1972. Unresolved problems in diagnosisand epidemiology of streptococcal infection, p. 567-570. In L. W. Wannamaker and J. M. Matsen (ed.),Streptococci and streptococcal diseases. AcademicPress Inc., New York.

11. Moody, M. D. 1972. Old and new techniques for rapididentification of group A streptococci, p. 177-188. InL. W. Wannamaker and J. M. Matsen (ed.), Strepto-cocci and streptococcal diseases. Academic Press Inc.,New York.

12. Murray, P. R., W. D. Wold, C. A. Schreck, and J. A.Washington. 1976. Effects of selective media and at-mosphere of incubation on the isolation of group Astreptococci. J. Clin. Microbiol. 4:54-56.

13. Nakamizo, Y., and M. Sato. 1972. New selective me-dium for the isolation of Streptococcus hemolyticus.Am. J. Clin. Pathol. 57:228-235.

14. Rosner, R. 1966. A new in vitro gram negative rodinhibiting agent which does not interfere with thegrowth of streptococci. Am. J. Med. Technol. 32:69-73.

15. Sanders, E. 1969. Bacterial interference. I. Its occur-rence among the respiratory tract flora and 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.Selective medium for the isolation of streptococcifrom clinical specimens. Appl. Microbiol. 22:942-943.

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