J Clin Pathol: first published as 10.1136/jcp.29.8.736 on 1 August 1976. Downloaded from J. clin. Path., 1976, 29, 736-739

Presumptive identification and antibiotic susceptibility of group B streptococci

A. M. M. JOKIPII AND LIISA JOKIPII From the Department of Serology and Bacteriology, University ofHelsinki, Helsinki, Finland

SYNOPSIS The comparative performance of three presumptive identification tests for group B haemolytic streptococci was investigated, using 371 different clinical isolates of group B strepto- cocci. Hippurate was hydrolysed by 96-1 %, the CAMP reaction was positive in 95 0 %, and pigment was produced by 97-3 %. A combination of any two tests would have detected over 99-8 %. On bile esculin agar 99 0% were able to grow, but none hydrolysed esculin; 51 % were susceptible to bacitracin. The minimum inhibitory concentrations of five antibiotics for 279 group B streptococci were determined. All were susceptible to penicillin G, ampicillin, cephalothin, and erythromycin, while 80 % were resistant to tetracycline. The MIC distributions were independent of the results of any identification test.

The impottance of group B fl-haemolytic strepto- esculin hydrolysis on bile esculin agar (Swan, 1954), cocci as human pathogens, especially during the characteristics of group A and group D streptococci,copyright. neonatal period, is well known (Duma et al, 1969; respectively. We also determined the in vitro suscepti- Eickhoff, 1972). Newborns acquire the organism bility of group B streptococci to five antibiotics. during passage through the birth canal; it has been suggested, therefore, that all pregnant women Material and methods should be screened for group B streptococci and, together with their husbands, treated if colonized by SELECTION OF STRAINS the organism (Franciosi et al, 1973). Screening for Over a period of 15 months, f-haemolytic strepto. group B streptococci may, therefore, be necessary for cocci, isolated in significant numbers from clinical http://jcp.bmj.com/ diagnostic and epidemiological purposes. Presump- specimens taken for routine bacteriological examina- tive identification schemes are required for this, tion, were grouped serologically with the technique since it may not be feasible in many clinical labora- of Swift et al (1943) using commercial antisera tories to group all streptococci serologically. (Behringwerke AG, Marburg-Lahn, Germany). Al- The ability to hydrolyse sodium hippurate (Ayers together 371 different isolates of group B strepto- and Rupp, 1922) and the production of the CAMP cocci were obtained; 166 of the specimens were factor (Christie, Atkins, and Munch-Petersen, 1944 from various departments of Helsinki University on October 2, 2021 by guest. Protected -from whom the name is derived), which lyses Central Hospital and 205 from general practitioners ruminant erythrocytes treated with staphylococcal in the Helsinki area. The strains were reported as ,-toxin, differentiate group B streptococci from most probable pathogens in the conditions from which other serological groups. Recently, pigment produc- they were isolated: vaginitis, Bartholinitis, ureth- tion in anaerobic conditions has been used as a ritis, , pharyngitis, rhinitis, sinusitis, distinctive characteristic of group B ,B-haemolytic otitis media, wound infection, or abscess (Eickhoff streptococci (Fallon, 1974). We have subjected fresh et al, 1964; Duma et al, 1969; Jelinkova et al, 1970; human isolates of group B haemolytic streptococci Eickhoff, 1972). to these identification tests to determine their performance in identifying group B streptococci in HIPPURATE HYDROLYSIS the practice of a clinical microbiological laboratory. The hydrolysis of sodium hippurate was determined In addition, they have been tested for susceptibility as described by Facklam et al (1974). The test strain to bacitracin (Maxted, 1953) and for growth and was inoculated into 5 ml of heart infusion broth (Difco, Detroit, Michigan) containing sodium hip- Received for publication 30 December 1975 purate, 10 mg/ml. After incubation at 35°C for 48 736 J Clin Pathol: first published as 10.1136/jcp.29.8.736 on 1 August 1976. Downloaded from

Presumptive identification and antibiotic susceptibility ofgroup B streptococci 737 hours, the cultures were centrifuged and the super- Test Number of Strains Positive natant was tested for benzoate with ferric chloride reagent. Positive Negative Hippurate hydrolysis 249 10 96-1 CAMP REACTION CAMP reaction 305 16 95 0 Pigment production 287 8 97 3 The production of CAMP factor was assayed by the Bacitracin susceptibility 16 300 5.1 modification of Barnum (1950) on 10% ox blood Bile esculin agar Growth 290 3 99 0 agar plates. A fl-toxin-producing staphylococcus Esculin hydrolysis 0 293 was inoculated as a diagonal on a plate, and the 00 streptococcal strain to be tested was streaked at Table I Reactions ofgroup B P-haemolytic right angles at a distance of 1-2 mm from the streptococci in presumptive identification tests staphylococcus. After overnight incubation at 35°C the plates were examined for synergistic haemolysis, the CAMP reaction. it failed to hydrolyse hippurate and was CAMP negative. PIGMENT PRODUCTION The frequency of bacitracin susceptibility was Test strains were inoculated on Columbia agar 5-1 %, the mean inhibition zone of the 16 strains plates (Oxoid, London). After overnight incubation being 13-3 mm. The theoretical numbers of baci- at 35°C anaerobically in the GasPak system, the tracin susceptible strains giving a negative result in presence or absence of reddish pigmentation of the one of the three tests for group B streptococci was colonies was recorded (Fallon, 1974). 0 5 for hippurate hydrolysis, 0-8 for CAMP re- activity, and 0'4 for pigment production. In fact, BILE ESCULIN AGAR one of the 16 bacitracin susceptible strains was hip- Test strains were inoculated on bile esculin agar purate negative, two were CAMP negative, and all produced pigment. plates (Swan, 1954), and the parameters recorded copyright. after overnight and after 40 hours' incubation at Esculin hydrolysis was not detected in any of the 35°C were growth and the production in and around 293 strains tested (table I), although all but three the colonies of a black colour, indicating esculin strains were able to grow on bile esculin plates, and hydrolysis. the growth was not essentially weaker than that of enterococci. The theoretical number of strains ANTIBIOTIC SUSCEPTIBILITY failing to grow on bile esculin and giving an excep- Paper discs containing 0-2 IU of bacitracin were tional result in one of the four other tests was 0 5, placed on densely streaked pure cultures of the and one such result was found. http://jcp.bmj.com/ streptococcal strains on blood agar plates, and the diameters of the inhibition zones were measured ANTIBIOTIC SUSCEPTIBILITY after overnight incubation at 35°C. The minimum All 279 different isolates of group B haemolytic inhibitory concentrations (MIC) of penicillin G, streptococci tested were susceptible to penicillin G, ampicillin, cephalothin, erythromycin, or tetra- ampicillin, cephalothin, and erythromycin (table II). cycline were determined with the plate dilution The MIC of tetracycline divided the strains into two major groups: 20% were inhibited by 0-8 ,tg/ml or method of MillIer-Hinton agar (Difco) containing on October 2, 2021 by guest. Protected 5 % horse blood using an inoculum-replicating de- less, 75% had a MIC of 50 ,ug/ml or higher, and only vice. The size of inoculum varied between 105 and 5% were intermediate. No significant deviations 106 colony-forming units and the results were read from the MIC distribution of the whole strain popu- after 24 hours' incubation at 35°C. lation were found within any of the five groups of strains that behaved abnormally in the presumptive Results identification tests, ie, within the hippurate negative, CAMP negative, pigment negative, bacitracin IDENTIFICATION TESTS susceptible, or bile susceptible groups. A summary of the results of tests on the 371 strains of group B haemolytic streptococci is shown in Discussion table I. Each of the three group B presumptive tests-hippurate hydrolysis, CAMP reactivity, and The 371 isolates of group B haemolytic streptococci pigment production-gave about the same propor- included in the survey were from a number of tion of positive results. The theoretical number of clinical conditions, in most of which group B strains giving a negative reaction in two tests streptococci have been reported previously. The simultaneously was 1-3. One such strain was found; present study was therefore confined to strains J Clin Pathol: first published as 10.1136/jcp.29.8.736 on 1 August 1976. Downloaded from

738 A. M. M. Jokipii and Liisa Jokipii

Antibiotic Minim4m Inhibitory Concentration (jug/ml) 0-05 0-2 0-8 3-1 12-5 50 > 50

Penicillin G 218 60 1 - - - Ampicillin 175 104 - Cephalothin 2 263 14 - - - Erythromycin 276 3 - - - - Tetracycline - 2 54 1 1 1 135 76 Table II Minimum inhibitory concentrations offive antibiotics for 279 isolates ofgroup B ,-haemolytic streptococci thought to be causing disease. These isolates were incubation and does not detect non-haemolytic subjected to presumptive identification tests not strains. The choice of the test(s) for screening groupB used in the selection of the strains; non-haemolytic streptococci can therefore be based on convenience group B streptococci were not included. rather than sensitivity. In the present study, the speci- Among the 259 group B streptococci tested, 10 ficity of the three presumptive tests in relation to did not hydrolyse sodium hippurate detectably. haemolytic streptococci of other Lancefield groups Thus the hippurate test would have detected 96-1 % was not investigated, but this may also guide the of the present strains. Using stock cultures, Ferrieri choice. Hippurate hydrolysis has been found with et al (1973) found hydrolysis of sodium hippurate in 6-9 % of group D enterococci, none of group A 100% of strains, and Facklam et al (1974) in 99-6 %. streptococci, and 0 3 % ofgroups other than A, B, or Our technique may have been less sensitive, since D(Facklam et al, 1974). Thus the test is expected tobe trace precipitations were regarded as negative. It is highly specific for group B streptococci, ifenterococci also possible that the strains used in the previous series can be excluded. Slightly over 20% of group A were selected, since the reports failed to indicate streptococci have been reported CAMP positivecopyright. whether presumptive tests had been performed be- (Heeschen et al, 1967; Lutticken and Fritsche, 1974). fore sending the streptococci to theculture collections. Fallon (1974) found that all pigment-producing Originally, the CAMP reaction was found to streptococci were of group B, which is also our ex- detect 100% of group B streptococci (Munch- perience so far, but we are not aware of systematic Petersen et al, 1945), but in later studies its sensitivity studies of the specificity of pigment production. Com- settled to an average of 98-4% (Barnum, 1950; parable figures of the specificity of the three tests are Esseveld et al, 1958; Biechteler, 1964; Pulverer, not available, and will be hard to obtain, mainly be- 1967; Heeschen et al, 1967), compared with 95-0% cause they are completely dependent on the distribu- http://jcp.bmj.com/ in the present investigation. tion of the various serological groups in the material Although pigment production, also, is not a new studied. observation, its use in the presumptive identification Growth on bile esculin agar without esculin of group B streptococci has received little attention. hydrolysis was present in 9900% of group B strains. Of 189 group B strains studied, Plummer (1941) Whether this property is sufficiently specific to be of found pigment production in 126 (67 %). Haemolysin any practical value in presumptive identification

was formed by 137 of the strains, but the possible should be further studied, since this test will often be on October 2, 2021 by guest. Protected association of haemolysin and pigment formation performed because of the resemblance of group B was not indicated. Fallon (1974) found pigment pro- and enterococcal colonies. duction in 86 % of 63 strains of group B haemolytic The in vitro antibiotic susceptibility of group B streptococci on primary under anaerobic streptococci was similar to that published more than conditions; none of the non-haemolytic group B 10 years ago (Eickhoff et al, 1964). All the 279 strains studied produced pigment. In the present strains studied were susceptible to penicillin G, series, 97 3 % of 295 strains of group B haemolytic ampicillin, cephalothin, and erythromycin, while streptococci produced pigment. most strains were resistant to tetracycline. Thus Negative results were infrequent in each of the routine susceptibility testing of group B isolates may three presumptive tests, and the simultaneous use of not be needed, although erythromycin resistant any two tests would have detected more than 99-8 % strains have been found previously (Eickhoff et al, of group B haemolytic streptococci. Each test has 1964; Jokipii and Jokipii, unpublished). features that may be inconvenient for the clinical laboratory: the hippurate test requires two days, the The authors are grateful for the excellent technical CAMP test is performed on sheep or bovine blood assistance of Mrs Anita Mutanen and for support agar, and pigment production requires anaerobic from the Paulo Foundation. J Clin Pathol: first published as 10.1136/jcp.29.8.736 on 1 August 1976. Downloaded from

Presumptive identification and antibiotic susceptibility ofgroup B streptococci 739 References group B haemolytic streptococci. J. clin. Path., 27, 902-905. Ferrieri, P., Wannamaker, L. W., and Nelson, J. (1973). Ayers, S. H. and Rupp, P. (1922). Differentiation of hemo- Localization and characterization of the hippuricase acti- lytic streptococci from human and bovine sources by the vity of group B streptococci. Infect. Immunol., 7, 747-752. hydrolysis of sodium hippurate. J. infect. Dis., 30, 388-399. Franciosi, R. A., Knostman, J. D., and Zimmerman, R. A. Barnum, D. A. (1950). The use of the CAMP test for the (1973). Group B streptococcal neonatal and infant infec- rapid identification of agalactiae. Rep. tions. J. Pediat., 82, 707-718. Ontario vet. Coll., pp. 120-125. Biechteler, W. (1964). Beitrag zur Routine-Diagnostik Heeschen, W., Tolle, A., and Zeidler, H. (1967). Zur Klassifi- hamolysierender Streptokokken (OBF-Test nach Guthoff, zierung der Gattung Streptococcus. Zbl. Bakt., I Abt. CAMP-Test, Antibiogramm). Zbl. Bakt., I Abt. Orig., Orig., 205, 250-259. 193, 48-56. Jelinkova, J., Neubauer, M., and Duben, J. (1970). Group B Christie, R., Atkins, N. E., and Munch-Petersen, E. (1944). streptococci in human pathology. Zbl. Bakt., I Abt. Orig., A note on a lytic phenomenon shown by group B strepto- 214, 450-457. cocci. Aust. J. exp. Biol. med. Sci., 22, 197-200. Liutticken, R. and Fritsche, D. (1974). Bestimmung der B- Duma, R. J., Weinberg, A. N., Medrek, T. F., and Kunz, Streptokokken () mit Hilfe des L. J. (1969). Streptococcal infections: a bacteriologic and "Dreifachtests" nach Wallerstrom und des CAMP-Tests. clinical study of streptococcal bacteremia. Medicine Zbl. Bakt., I Abt. Orig., 226, 298-304. (Baltimore), 48, 87-127. Maxted, W. R. (1953). The use of bacitracin for identifying Eickhoff, T. C. (1972). Group B streptococci in human infec- group A haemolytic streptococci. J. clin. Path., 6, 224-226. tion. In Streptococci and Streptococcal Diseases, edited by Munch-Petersen, E., Christie, R., and Simmons, R. T. (1945). L. W. Wannamaker, and J. M. Matsen, pp. 533-541. Further notes on a lytic phenomenon shown by group B Academic Press, New York. streptococci. Aust. J. exp. Biol. med. Sci., 23, 193-195. Eickhoff, T. C., Klein, J. O., Daly, A. K., Ingall, D., and Plummer, H. (1941). A serological and biochemical study of Finland, M. (1964). Neonatal sepsis and other infections hemolytic streptococci. J. Immunol., 42, 91-107. due to group B beta-hemolytic streptococci. New Engl. J. Pulverer, G. (1967). Gruppendifferenzierung beta-hamo- Med., 271, 1221-1228. lysierender Streptokokken mit Hilfe des OBF-Testes nach Esseveld, H., Daniels-Bosman, M. S. M., and Leijnse, B. Guthof und des CAMP-Testes. Zbl. Bakt., I Abt. Orig., (1958). Some observations about the CAMP reaction and 204, 301-304. its application to human ,-haemolytic streptococci. Swan, A. (1954). The use of a bile-aesculin medium and of Antonie v. Leeuwenhoek, 24, 145-156. Maxted's technique of in the identifi- Facklam, R. R., Padula, J. F., Thacker, L. G., Wortham, cation of enterococci (group D streptococci). J. clin. Path., copyright. E. C., and Sconyers, B. J. (1974). Presumptive identifica- 7, 160-163. tion of group A, B, and D streptococci. Appl. Microbiol., Swift, H. F., Wilson, A. T., and Lancefield, R. C. (1943). 27, 107-113. Typing group A hemolytic streptococci by M precipitin Fallon, R. J. (1974). The rapid recognition of Lancefield reactions in capillary pipettes. J. exp. Med., 78, 127-133. http://jcp.bmj.com/ on October 2, 2021 by guest. Protected