Zbl. Bakt. 277, 260-266 (1992) © Gustav Fischer Verlag, Stuttgart/New York
Serotyping and Further Characterization of Group B Streptococcal Isolates from Indonesia I. W. T . WIBAWAW , CH . LAMMLER 1 .
,
Y. LAUTROU 1 + ,
and U . CH . WARSA 2 1
2
Institut fur Bakteriologie und Immunologie der justus-Liebig-Universitar Giel.sen, 6300 Giel.sen, Germany Department of Microbiology, Medical Faculty, University of Indonesia, Jakarta 10320, Ind onesia
With 1 Figure· Received November 14, 1991 . Revision received January 10, 1992 . Accepted February 24, 1992
Summary Serotyping of 103 streptococci of sero logical group B isolated from human clinical specimen s in Indonesia revealed mainl y type pattern laic and type patterns III and V, either alone or in combination with protein antigens c or R. Few cultures had serotype II and IV, none of the cultur es had type antigen Ib or X. Extracts of one group B streptococcal culture reacted with antibodies specific to type antigens II, IV and R and one culture with antibodies specific to candida te type 727 1. Protein antigen c was present as en, ca, ~ but not as c~ component . Group B streptococci with type antigen R grew generally as a granular sediment with clear supern atant, formed co mpact colonies in soft agar and reacted positively in the salt aggregation test . Group B streptococci without surfa ce antigen R grew mainly with a uniform turbidity of the growth medi a, fo rmed diffuse colonies in soft agar and were negative in the salt aggregation test.
Zusammenfassung Die Serotypisierung von 103 Streptokokkenkulturen der serologischen Gruppe B, isoliert in Indonesien aus Untersuchungsmaterial vom Menschen, ergab hauptsachlich die Antigentypen laIc, III und V allein oder in Kombination mit den Proteinantigenen c und R. Einige Kulturen hatten die Typenantigene II und IV, bei keiner der Kulturen w aren die Typenantigene Ib oder X nachweisbar. Die Antigenpraparation einer B-Streptokokkenkultur reagierte mit Typenantigen II, IV und R spezifischen Antikorpern, die Ant igenpraparation einer weiteren Kultur mit spezifischen Antikorpern gegen den Typenkandidaten 72 71. Das Protein antigen c konnte mit seiner ca Komponenten allein, beiden Komponenten ca, c~ zusammen, nicht dagegen mit seiner c~ Komponenten allein nachgewiesen werden.
• Co rresponding author + Co llabo rator via fellow ship under OE CD Project on Biological Resource Management
Serotyping and Further Characterization of Group B Streptococcal Isolates
261
Die B-Streptokokkenkulturen mit Typenantigen R wuchsen iiberwiegend als granulates Sediment mit klarem Uberstand, bildeten kompakte Kolonien in Soft Agar Medium und reagierten positiv im Salzaggregationstest. B-Streptokokkenkulturen ohne Oberflachenantigen R wuchsen iiberwiegend mit einer gleichforrnigen Triibung des Wachstumsmediums, bildeten diffuse Kolonien in Soft Agar Medium und zeigten keine Reaktionen im Salzaggregationstest.
Introduction Streptococci of serological group B play an etiological role both in bovine mastitis and in human neonatal infections, particularly in septicemia and meningitis (6, 10). The human neonate infections generally occur as an early-onset disease within the first five days of life resulting from acquisition of the group B streptococci in the uterus or during the passage through the birth canal. Vaginal colonization in the mother provides the major reservoir of group B streptococci from which neonatal infections derive during parturition (1, 5). Group B streptococcal infections in older infants, termed lateonset disease, are associated with severe neurological sequelae. Though less common, other infectious processes caused by group B streptococci have been reported (4, 14). Current knowledge of the epidemiology of group B streptococcal diseases in humans and the relation between infections of humans and bovines has been obtained by serotyping the organism. Recent studies of group B streptococcal isolates obtained from human specimens in Germany revealed mainly the serotype patterns laic and III/R (11). The occurrence of type antigen R as well as type antigen X has been shown to be related to specific growth characteristics of the bacteria and seemed to influence the group B streptococcal behaviour in the salt aggregation test (12). The present study was performed on serotype group B streptococcal isolates obtained from human clinical specimens in Indonesia. The typing results were compared with further characteristics of the bacteria.
Materials and Methods Bacterial cultures. A total of 103 group B streptococci isolated from humans were used in this study. The cultures were collected from routine specimens at the Department of Microbiology in Jakarta, Indonesia. The cultures were isolated from urine, infected skin, sputum, sperm and bronchial washings. Serogrouping was performed with autoclaved extracts and group B streptococcal antisera (Wellcome, Burgwedel, Germany) in immunodiffusion reactions. The cultures were additionally tested for CAMP reactivity (8) and pigment production. The latter was performed in GBS Islam agar (Oxoid, Wesel, Germany) supplemented with 50 ml sterile horse serum/l as stab cultures in screw-capped tubes. The cultures were maintained on sheep blood agar and subcultured every 4 weeks. For growth in fluid media, the bacteria were cultivated in Todd-Hewitt Broth (THB, GIBCO, Europe, Karlsruhe, Germany) for 24 h at 3rc. Serotyping. For serotyping, the type-specific antigens were obtained by extraction of the bacteria in 0.2 M HCl for 2 h 52°C. The antigens were tested with monospecific antibodies in coagglutination and immunodiffusion reactions as described previously (11). The monospecific antibodies were prepared with the B streptococcal reference strains 090 (Ia), H 36 B (Ib), 18 RS 21 (II), 6313 (III), 3139 (IV), SS 1169 (V), A 909 (Ie), 25/60 (R), 24/60 (X), 335 (co), 70339 (c~), NT 6 and 7271.
262
I. W.T. Wibawan, Ch. Lammler, Y.Lautrou, and U. Ch. Warsa
Soft agar technique. For this, the group B streptococci were cultivated in brain heart infusion (BHI, GIBCO), containing 0.15% agar as described previously (13). After 18 hat 3rC, growth of the bacteria was examined for compact or diffuse colony formation. Salt aggregation test (SAT). SAT was performed with photometrically adjusted bacteria (109 cells/ml) and equal volumes of 2 M ammonium sulphate on microscopic slides (12).
Results All 103 cultures used in the present investigation reacted with group B-specific antisera, produced a hazy to clear zone of ~-hemolysis and gave a positive CAMP reaction in the zone of staphylococcal beta lysin. In GBS Islam agar, 101 of the cultures were clearly pigmented. The available monospecific antibodies allowed a serotyping of 81% of the group B streptococcal cultures. Identical results were obtained by using the coagglutination and immunodiffusion reactions. The type patterns of the 103 group B streptococci are summarized in Tables 1 and 2. Among the cultures, 15 (14%) had the antigen pattern la/c, 9 cultures (8%) IIIIR, 8 cultures (8%) type III and type V, respectively, and 7 cultures (7%) type IV, type VIR and type NT/c, respectively. The remaining cultures had the antigen pattern II (6 cultures-6%), IIIIc (5 cultures-5%), la (4 cultures-4%), NT/R (3 cultures-3°/o), II1R (2 cultures-2%), II1c (1 culture-I %) and Vic (1 culture-1 %). One culture with type pattern II1R additionally reacted with type-IVspecific antibodies and one non-typable culture with specific antibodies against candidate type 7271 (Fig. 1). None of the cultures reacted with specific antibodies against type antigen Ib, X and candidate type NT 6, 20 cultures (19%) were non-typable (Table 1). Of 29 group B streptococcal cultures with protein antigen c, 12 cultures (41%) had antigen ca and 17 cultures (59%) had both antigenic components ca and c~. None of the cultures had the c~ component alone (Table 2). After cultivation of the bacteria in fluid media, the group B streptococci could be differentiated by their turbidity patterns. Group B streptococci with type antigen R grew mainly as a granular sediment with clear supernatant, those without antigen R with a uniform turbidity of the growth media. Among the non-typable group B streptococci, 11 cultures grew as granular sediment and 9 cultures with a uniform turbidity of the media. In addition, cultures with type antigen R generally formed compact colonies in soft agar. Those without antigen R mainly formed a diffuse type of colony morphology. Of the non-typable cultures, 13 exhibited a compact and 7, a diffuse colony morphology in soft agar. In the SAT group B streptococci with type antigen R
Table 1. Type antigen patterns of 103 group B streptococcal cultures isolated from humans Protein antigens c R
z 'f
Polysaccharide antigens Ia 4" 15
Ib
II
III
IV
V
NT
~
6 1
8 5 9 22
7
8 1 7 16
20** 7 3 30
53 29 21 103
2
19
9
7
,. Number of cultures with the respective antigen pattern. * One culture demonstrates a reaction with specificantibodies against candiate type 7271.
Serotyping and Further Characterization of Gro up B Streptococcal Isolates Table 2. Distribution of type antigens cc and pro tein type antige n c
c~
263
among group B streptococcal cultures with
Polysaccharide ant igens Antigen c
Ia
Ct Ct,B L
4* 11 15
lb
II
1Il
IV
V
3 2 5
NT
L
4
12 17 29
3
1 1
7
* See Table 1.
Fig. 1. Specific reaction of hot acid extracts of group B streptococcal cultures 7271 (a) and B Jakarta 61A (b) with purified specific antibodies against candidate type 7271 (1).
Table 3. Reactions of 103 group B streptococci with and without surface antigen R No. of cultures with result in testa Culture and antigen (n)
Growth T 5
Soft agar D C
+
R (21) No anti~en R (62) NT (20)
17 4 11
17 6 13
14 17 12
4 58 9
4 56 7
SAT 7 45 8
a S = Grow th as sediment with clear supernatant , T = Uniformly turbid growth, C = Com pact growth in soft agar, D = Diffuse growth in soft agar, SAT = Reaction in salt aggregation test, p < 0.001 for all results (modified Fisher test). b NT = non typa ble.
were mainly positive and those without antigen R, mainly nega tive. Of the non-typable cu ltures, 12 were positive and 8 we re negative in the SAT (Table 3). Cultures with type antigen R generally grew as granular sediment, showed compact growth in soft agar media and reacted posi tively in the SAT . Those without antigen R generally grew w ith an uniform turbidity of the fluid media, had diffuse colonies in soft agar and were negative in the SAT. Statistical ana lysis revealed the high significance of this relations (T able 4, 5 ).
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I. W. T. Wibawan, Ch. Lammler, Y.Lautrou, and U. Ch. Warsa
Table 4. Relationship between culture growth and other test results No. of cultures with result in testa Growth pattern of culture Sediment Turbid
Soft agar
SAT
C
D
+
29
3 64
26
7
6 54
17
p < 0.001 for all results (modified Fisher test). a See table 3.
Table 5. Relationship between soft agar growth and SAT results Soft agar growth of cultures
No. of cultures with SAT results
+
Compact Diffuse
26
10
17
50
p < 0.001 for all results (modified Fisher test).
Table 6. Relationship between culture growth of 20 non-rypable cultures (NT) of group B streptococci and other test results No. of cultures with result in test" Growth pattern of culture
Soft agar C D
Sediment Turbid
11 2
o 7
SAT
+ 10 2
1 7
a See table 3.
Table 7. Relationship between soft agar growth of 20 non-typable cultures (NT) and SAT results Soft agar growth of cultures Compact Diffuse
No. of cultures with SAT results
+ 10 2
3 5
Serotyping and Further Characterization of Group B StreptococcalIsolates
265
A comparable relation could be observed among the non-typable cultures. Cultures which grew as granular sediment were generally compact in soft agar and SAT-positive. Those with uniform turbidity of growth media were generally diffuse in soft agar and SAT-negative, Because of the low number of culrures the non-typable cultures were not included in the statistical analyses (Table 6, 7).
Discussion Polysaccharide and protein surface antigens allow a further characterization of streptococci of serological group B. This has proved to be useful for epidemiological studies. However, up to date no information is available about serotypes of Indonesian group B streptococci. In the present study, recently purified monospecific antibodies against group B streptococcal polysaccharide and protein type antigens were used to serotype group B streptococcal isolates from Indonesia. The occurrence of type antigens was compared with further characteristics of the bacteria. The serotyping revealed that most of the cultures had serotype laic, serotype III and the newly described serotype V, either alone or in combination with protein antigen R or c. Part of the cultures had serotype II and the newly described serotype IV. These results corresponded to previous findings on German group B streptococcal isolates (11). It was of interest that again none of the cultures had type antigen Ib or X. Type antigen X seems to be related to bovine pathogenic group B streptococci and may also occur among streptococci of serological groups G and L (2,9, 11). Antigen preparations of one of the cultures reacted with antibodies specific to polysaccharide types II and IV. To ensure that the extracts were not obtained from a mixture of two cultures the antigens were prepared starting from a single colony. The occurrence of two polysaccharide antigens on the surface of group B streptococci has also been described by ]elinkova et al. (7). The extracts of one of the non-typable group B streptococcal cultures reacted with specific antibodies against candidate type 7271. Comparable reactions with antibodies specific to candidate type 7271 have been described previously (11). This may again contribute to establish strain 7271 as a new internationally used type reference strain. A further differentiation of cc and c~ components among cultures with protein antigen c has revealed that the ca component occurs either alone or in combination with c~. None of the cultures had the c~ component alone. The rarity of the occurrence of the c~ component alone had been described earlier (3, 11). After cultivation of the group B streptococci in fluid media, those with surface antigen R grew as a granular sediment with clear supernatant. Additionally, the cultures showed compact colony formation in soft agar and reacted positively in the SAT. Those without surface antigen R mainly grew with a uniform turbidity of the growth media, had diffuse colonies in soft agar and were negative in the SAT. These properties, possibly related to the surface charge of the bacteria have been described earlier for "human" and "bovine" group B streptococci with and without surface antigens Rand X, respectively (12). The latter characteristics also divide the non-typable group B streptococci into two groups. One group of non-typable group B streptococci with growth in fluid media as granular sediment, compact colony formation in soft agar and a positive SAT reaction may possess a hitherto not described protein surface antigen. Those exhibiting an uniform turbidity of the growth media, diffuse colonies in soft agar and a negative SAT
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I. W. T. Wibawan, Ch. Lammler, Y.Lautrou, and U. Ch. Warsa
reaction might represent a group with a hitherto unknown polysaccharide type antigen. Together with growth characteristics in fluid media and soft agar and the behaviour of the bacteria in SAT, the determination of specific serotypes could be used for individual characterization of group B streptococcal isolates. This may help to clarify epidemiological problems.
References 1. Anthony, B. W., R. Eisenstadt, j. Carter, K. S. Kim, and C. j. Hobel: Genital and intestinal carriage of group B streptococci during pregnancy. J. Infect. Dis. 143 (1981) 761-766 2. Bergner-Rabinowitz, S., M. Ferne, S. Fleidermann, G. Ziv, A. Saran, and M. Winkler: Group G type X: a new antigenic combination in streptococci isolated from cases of bovine mastitis in Israel. Vet. Microbiol. 6 (1981) 383-387 3. Bevanger, L.: Ibc proteins as serotype markers of group B streptococci. Acta path. microbiol. immunol. scand. Sect. B 91 (1983) 231-234 4. Gallagher, P. G. and C. Watanakunakorn: Group B streptococcal endocarditis: Report of seven cases and review of the literature, 1962-1985. Rev. Infect. Dis. 8 (1986) 175-188 5. Hastings, M. j. G., C. S. F. Easmon, j. Neill, B. Bloxham, and R. P. A. Rivers: Group B streptococcal colonization and the outcome of pregnancy. J. Infect. 12 (1986) 23-29 6. jelinkova, j.: Group B streptococci in the human population. Curr. Top. Microbiol. Immunol. 76 (1977) 127-165 7. jelinkova, j., M. Wagner, j. Motlova, B. Wagner, N. E. jensen, and V. Kubin: Immunoelectron microscopic demonstration of antigenic structures of group B streptococci. XIth Lancefield International Symposium on Streptococci and Streptococcal Diseases, 10.-14.09.1990, Siena/Italy, Abstract L 64 (1990) 8. Lammler, C. and H. Blobel: Synergistische und antagonistische hamolytische Reaktionen bakterieller Proteine. Bed. Munch. Tierarzrl. Wschr. 100 (1987) 95-99 9. Ldmmler, C, K. Giirtiirk, and H. Blobel: Streptococcal group B type antigen X in group L streptococci. J. Clin. Microbiol. 25 (1987) 383-387 10. Ross, P. W.: Group B streptococcus-profile of an organism. J. Med. Microbiol. 18 (1984) 139-166 11. Wibawan, I. W. T. and Ch. Ldmmler: Distribution of B streptococcal type antigens among streptococci of serological groups B, G and L. Zbl. Bakt. 273 (1990) 471-477 12. Wibawan, I. W. T. and Ch. Lammler: Properties of group B streptococci with protein surface antigens X and R. ]. Clin. Microbiol. 28 (1990) 2834-2836 13. Wibawan, I. W. T. and Ch. Ldmmler: Demonstration of a and ~ components of group B streptococcal protein antigen c in serum-soft agar. J. Vet. Med. B 37 (1990) 680-683 14. Wilkinson, H. W.: Group B streptococcal infection in humans. Annu. Rev. Microbiol. 32 (1978) 41-57 Dr. Christoph Lammler, Institut fur Bakteriologie und Immunologie der ]ustus-LiebigUniversitat, Frankfurter Str. 107, D-6300 GielSen
Serotyping and Further Characterization of Group B Streptococcal Isolates from Indonesia I. W. T . WIBAWAW , CH . LAMMLER 1 .
,
Y. LAUTROU 1 + ,
and U . CH . WARSA 2 1
2
Institut fur Bakteriologie und Immunologie der justus-Liebig-Universitar Giel.sen, 6300 Giel.sen, Germany Department of Microbiology, Medical Faculty, University of Indonesia, Jakarta 10320, Ind onesia
With 1 Figure· Received November 14, 1991 . Revision received January 10, 1992 . Accepted February 24, 1992
Summary Serotyping of 103 streptococci of sero logical group B isolated from human clinical specimen s in Indonesia revealed mainl y type pattern laic and type patterns III and V, either alone or in combination with protein antigens c or R. Few cultures had serotype II and IV, none of the cultur es had type antigen Ib or X. Extracts of one group B streptococcal culture reacted with antibodies specific to type antigens II, IV and R and one culture with antibodies specific to candida te type 727 1. Protein antigen c was present as en, ca, ~ but not as c~ component . Group B streptococci with type antigen R grew generally as a granular sediment with clear supern atant, formed co mpact colonies in soft agar and reacted positively in the salt aggregation test . Group B streptococci without surfa ce antigen R grew mainly with a uniform turbidity of the growth medi a, fo rmed diffuse colonies in soft agar and were negative in the salt aggregation test.
Zusammenfassung Die Serotypisierung von 103 Streptokokkenkulturen der serologischen Gruppe B, isoliert in Indonesien aus Untersuchungsmaterial vom Menschen, ergab hauptsachlich die Antigentypen laIc, III und V allein oder in Kombination mit den Proteinantigenen c und R. Einige Kulturen hatten die Typenantigene II und IV, bei keiner der Kulturen w aren die Typenantigene Ib oder X nachweisbar. Die Antigenpraparation einer B-Streptokokkenkultur reagierte mit Typenantigen II, IV und R spezifischen Antikorpern, die Ant igenpraparation einer weiteren Kultur mit spezifischen Antikorpern gegen den Typenkandidaten 72 71. Das Protein antigen c konnte mit seiner ca Komponenten allein, beiden Komponenten ca, c~ zusammen, nicht dagegen mit seiner c~ Komponenten allein nachgewiesen werden.
• Co rresponding author + Co llabo rator via fellow ship under OE CD Project on Biological Resource Management
Serotyping and Further Characterization of Group B Streptococcal Isolates
261
Die B-Streptokokkenkulturen mit Typenantigen R wuchsen iiberwiegend als granulates Sediment mit klarem Uberstand, bildeten kompakte Kolonien in Soft Agar Medium und reagierten positiv im Salzaggregationstest. B-Streptokokkenkulturen ohne Oberflachenantigen R wuchsen iiberwiegend mit einer gleichforrnigen Triibung des Wachstumsmediums, bildeten diffuse Kolonien in Soft Agar Medium und zeigten keine Reaktionen im Salzaggregationstest.
Introduction Streptococci of serological group B play an etiological role both in bovine mastitis and in human neonatal infections, particularly in septicemia and meningitis (6, 10). The human neonate infections generally occur as an early-onset disease within the first five days of life resulting from acquisition of the group B streptococci in the uterus or during the passage through the birth canal. Vaginal colonization in the mother provides the major reservoir of group B streptococci from which neonatal infections derive during parturition (1, 5). Group B streptococcal infections in older infants, termed lateonset disease, are associated with severe neurological sequelae. Though less common, other infectious processes caused by group B streptococci have been reported (4, 14). Current knowledge of the epidemiology of group B streptococcal diseases in humans and the relation between infections of humans and bovines has been obtained by serotyping the organism. Recent studies of group B streptococcal isolates obtained from human specimens in Germany revealed mainly the serotype patterns laic and III/R (11). The occurrence of type antigen R as well as type antigen X has been shown to be related to specific growth characteristics of the bacteria and seemed to influence the group B streptococcal behaviour in the salt aggregation test (12). The present study was performed on serotype group B streptococcal isolates obtained from human clinical specimens in Indonesia. The typing results were compared with further characteristics of the bacteria.
Materials and Methods Bacterial cultures. A total of 103 group B streptococci isolated from humans were used in this study. The cultures were collected from routine specimens at the Department of Microbiology in Jakarta, Indonesia. The cultures were isolated from urine, infected skin, sputum, sperm and bronchial washings. Serogrouping was performed with autoclaved extracts and group B streptococcal antisera (Wellcome, Burgwedel, Germany) in immunodiffusion reactions. The cultures were additionally tested for CAMP reactivity (8) and pigment production. The latter was performed in GBS Islam agar (Oxoid, Wesel, Germany) supplemented with 50 ml sterile horse serum/l as stab cultures in screw-capped tubes. The cultures were maintained on sheep blood agar and subcultured every 4 weeks. For growth in fluid media, the bacteria were cultivated in Todd-Hewitt Broth (THB, GIBCO, Europe, Karlsruhe, Germany) for 24 h at 3rc. Serotyping. For serotyping, the type-specific antigens were obtained by extraction of the bacteria in 0.2 M HCl for 2 h 52°C. The antigens were tested with monospecific antibodies in coagglutination and immunodiffusion reactions as described previously (11). The monospecific antibodies were prepared with the B streptococcal reference strains 090 (Ia), H 36 B (Ib), 18 RS 21 (II), 6313 (III), 3139 (IV), SS 1169 (V), A 909 (Ie), 25/60 (R), 24/60 (X), 335 (co), 70339 (c~), NT 6 and 7271.
262
I. W.T. Wibawan, Ch. Lammler, Y.Lautrou, and U. Ch. Warsa
Soft agar technique. For this, the group B streptococci were cultivated in brain heart infusion (BHI, GIBCO), containing 0.15% agar as described previously (13). After 18 hat 3rC, growth of the bacteria was examined for compact or diffuse colony formation. Salt aggregation test (SAT). SAT was performed with photometrically adjusted bacteria (109 cells/ml) and equal volumes of 2 M ammonium sulphate on microscopic slides (12).
Results All 103 cultures used in the present investigation reacted with group B-specific antisera, produced a hazy to clear zone of ~-hemolysis and gave a positive CAMP reaction in the zone of staphylococcal beta lysin. In GBS Islam agar, 101 of the cultures were clearly pigmented. The available monospecific antibodies allowed a serotyping of 81% of the group B streptococcal cultures. Identical results were obtained by using the coagglutination and immunodiffusion reactions. The type patterns of the 103 group B streptococci are summarized in Tables 1 and 2. Among the cultures, 15 (14%) had the antigen pattern la/c, 9 cultures (8%) IIIIR, 8 cultures (8%) type III and type V, respectively, and 7 cultures (7%) type IV, type VIR and type NT/c, respectively. The remaining cultures had the antigen pattern II (6 cultures-6%), IIIIc (5 cultures-5%), la (4 cultures-4%), NT/R (3 cultures-3°/o), II1R (2 cultures-2%), II1c (1 culture-I %) and Vic (1 culture-1 %). One culture with type pattern II1R additionally reacted with type-IVspecific antibodies and one non-typable culture with specific antibodies against candidate type 7271 (Fig. 1). None of the cultures reacted with specific antibodies against type antigen Ib, X and candidate type NT 6, 20 cultures (19%) were non-typable (Table 1). Of 29 group B streptococcal cultures with protein antigen c, 12 cultures (41%) had antigen ca and 17 cultures (59%) had both antigenic components ca and c~. None of the cultures had the c~ component alone (Table 2). After cultivation of the bacteria in fluid media, the group B streptococci could be differentiated by their turbidity patterns. Group B streptococci with type antigen R grew mainly as a granular sediment with clear supernatant, those without antigen R with a uniform turbidity of the growth media. Among the non-typable group B streptococci, 11 cultures grew as granular sediment and 9 cultures with a uniform turbidity of the media. In addition, cultures with type antigen R generally formed compact colonies in soft agar. Those without antigen R mainly formed a diffuse type of colony morphology. Of the non-typable cultures, 13 exhibited a compact and 7, a diffuse colony morphology in soft agar. In the SAT group B streptococci with type antigen R
Table 1. Type antigen patterns of 103 group B streptococcal cultures isolated from humans Protein antigens c R
z 'f
Polysaccharide antigens Ia 4" 15
Ib
II
III
IV
V
NT
~
6 1
8 5 9 22
7
8 1 7 16
20** 7 3 30
53 29 21 103
2
19
9
7
,. Number of cultures with the respective antigen pattern. * One culture demonstrates a reaction with specificantibodies against candiate type 7271.
Serotyping and Further Characterization of Gro up B Streptococcal Isolates Table 2. Distribution of type antigens cc and pro tein type antige n c
c~
263
among group B streptococcal cultures with
Polysaccharide ant igens Antigen c
Ia
Ct Ct,B L
4* 11 15
lb
II
1Il
IV
V
3 2 5
NT
L
4
12 17 29
3
1 1
7
* See Table 1.
Fig. 1. Specific reaction of hot acid extracts of group B streptococcal cultures 7271 (a) and B Jakarta 61A (b) with purified specific antibodies against candidate type 7271 (1).
Table 3. Reactions of 103 group B streptococci with and without surface antigen R No. of cultures with result in testa Culture and antigen (n)
Growth T 5
Soft agar D C
+
R (21) No anti~en R (62) NT (20)
17 4 11
17 6 13
14 17 12
4 58 9
4 56 7
SAT 7 45 8
a S = Grow th as sediment with clear supernatant , T = Uniformly turbid growth, C = Com pact growth in soft agar, D = Diffuse growth in soft agar, SAT = Reaction in salt aggregation test, p < 0.001 for all results (modified Fisher test). b NT = non typa ble.
were mainly positive and those without antigen R, mainly nega tive. Of the non-typable cu ltures, 12 were positive and 8 we re negative in the SAT (Table 3). Cultures with type antigen R generally grew as granular sediment, showed compact growth in soft agar media and reacted posi tively in the SAT . Those without antigen R generally grew w ith an uniform turbidity of the fluid media, had diffuse colonies in soft agar and were negative in the SAT. Statistical ana lysis revealed the high significance of this relations (T able 4, 5 ).
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I. W. T. Wibawan, Ch. Lammler, Y.Lautrou, and U. Ch. Warsa
Table 4. Relationship between culture growth and other test results No. of cultures with result in testa Growth pattern of culture Sediment Turbid
Soft agar
SAT
C
D
+
29
3 64
26
7
6 54
17
p < 0.001 for all results (modified Fisher test). a See table 3.
Table 5. Relationship between soft agar growth and SAT results Soft agar growth of cultures
No. of cultures with SAT results
+
Compact Diffuse
26
10
17
50
p < 0.001 for all results (modified Fisher test).
Table 6. Relationship between culture growth of 20 non-rypable cultures (NT) of group B streptococci and other test results No. of cultures with result in test" Growth pattern of culture
Soft agar C D
Sediment Turbid
11 2
o 7
SAT
+ 10 2
1 7
a See table 3.
Table 7. Relationship between soft agar growth of 20 non-typable cultures (NT) and SAT results Soft agar growth of cultures Compact Diffuse
No. of cultures with SAT results
+ 10 2
3 5
Serotyping and Further Characterization of Group B StreptococcalIsolates
265
A comparable relation could be observed among the non-typable cultures. Cultures which grew as granular sediment were generally compact in soft agar and SAT-positive. Those with uniform turbidity of growth media were generally diffuse in soft agar and SAT-negative, Because of the low number of culrures the non-typable cultures were not included in the statistical analyses (Table 6, 7).
Discussion Polysaccharide and protein surface antigens allow a further characterization of streptococci of serological group B. This has proved to be useful for epidemiological studies. However, up to date no information is available about serotypes of Indonesian group B streptococci. In the present study, recently purified monospecific antibodies against group B streptococcal polysaccharide and protein type antigens were used to serotype group B streptococcal isolates from Indonesia. The occurrence of type antigens was compared with further characteristics of the bacteria. The serotyping revealed that most of the cultures had serotype laic, serotype III and the newly described serotype V, either alone or in combination with protein antigen R or c. Part of the cultures had serotype II and the newly described serotype IV. These results corresponded to previous findings on German group B streptococcal isolates (11). It was of interest that again none of the cultures had type antigen Ib or X. Type antigen X seems to be related to bovine pathogenic group B streptococci and may also occur among streptococci of serological groups G and L (2,9, 11). Antigen preparations of one of the cultures reacted with antibodies specific to polysaccharide types II and IV. To ensure that the extracts were not obtained from a mixture of two cultures the antigens were prepared starting from a single colony. The occurrence of two polysaccharide antigens on the surface of group B streptococci has also been described by ]elinkova et al. (7). The extracts of one of the non-typable group B streptococcal cultures reacted with specific antibodies against candidate type 7271. Comparable reactions with antibodies specific to candidate type 7271 have been described previously (11). This may again contribute to establish strain 7271 as a new internationally used type reference strain. A further differentiation of cc and c~ components among cultures with protein antigen c has revealed that the ca component occurs either alone or in combination with c~. None of the cultures had the c~ component alone. The rarity of the occurrence of the c~ component alone had been described earlier (3, 11). After cultivation of the group B streptococci in fluid media, those with surface antigen R grew as a granular sediment with clear supernatant. Additionally, the cultures showed compact colony formation in soft agar and reacted positively in the SAT. Those without surface antigen R mainly grew with a uniform turbidity of the growth media, had diffuse colonies in soft agar and were negative in the SAT. These properties, possibly related to the surface charge of the bacteria have been described earlier for "human" and "bovine" group B streptococci with and without surface antigens Rand X, respectively (12). The latter characteristics also divide the non-typable group B streptococci into two groups. One group of non-typable group B streptococci with growth in fluid media as granular sediment, compact colony formation in soft agar and a positive SAT reaction may possess a hitherto not described protein surface antigen. Those exhibiting an uniform turbidity of the growth media, diffuse colonies in soft agar and a negative SAT
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reaction might represent a group with a hitherto unknown polysaccharide type antigen. Together with growth characteristics in fluid media and soft agar and the behaviour of the bacteria in SAT, the determination of specific serotypes could be used for individual characterization of group B streptococcal isolates. This may help to clarify epidemiological problems.
References 1. Anthony, B. W., R. Eisenstadt, j. Carter, K. S. Kim, and C. j. Hobel: Genital and intestinal carriage of group B streptococci during pregnancy. J. Infect. Dis. 143 (1981) 761-766 2. Bergner-Rabinowitz, S., M. Ferne, S. Fleidermann, G. Ziv, A. Saran, and M. Winkler: Group G type X: a new antigenic combination in streptococci isolated from cases of bovine mastitis in Israel. Vet. Microbiol. 6 (1981) 383-387 3. Bevanger, L.: Ibc proteins as serotype markers of group B streptococci. Acta path. microbiol. immunol. scand. Sect. B 91 (1983) 231-234 4. Gallagher, P. G. and C. Watanakunakorn: Group B streptococcal endocarditis: Report of seven cases and review of the literature, 1962-1985. Rev. Infect. Dis. 8 (1986) 175-188 5. Hastings, M. j. G., C. S. F. Easmon, j. Neill, B. Bloxham, and R. P. A. Rivers: Group B streptococcal colonization and the outcome of pregnancy. J. Infect. 12 (1986) 23-29 6. jelinkova, j.: Group B streptococci in the human population. Curr. Top. Microbiol. Immunol. 76 (1977) 127-165 7. jelinkova, j., M. Wagner, j. Motlova, B. Wagner, N. E. jensen, and V. Kubin: Immunoelectron microscopic demonstration of antigenic structures of group B streptococci. XIth Lancefield International Symposium on Streptococci and Streptococcal Diseases, 10.-14.09.1990, Siena/Italy, Abstract L 64 (1990) 8. Lammler, C. and H. Blobel: Synergistische und antagonistische hamolytische Reaktionen bakterieller Proteine. Bed. Munch. Tierarzrl. Wschr. 100 (1987) 95-99 9. Ldmmler, C, K. Giirtiirk, and H. Blobel: Streptococcal group B type antigen X in group L streptococci. J. Clin. Microbiol. 25 (1987) 383-387 10. Ross, P. W.: Group B streptococcus-profile of an organism. J. Med. Microbiol. 18 (1984) 139-166 11. Wibawan, I. W. T. and Ch. Ldmmler: Distribution of B streptococcal type antigens among streptococci of serological groups B, G and L. Zbl. Bakt. 273 (1990) 471-477 12. Wibawan, I. W. T. and Ch. Lammler: Properties of group B streptococci with protein surface antigens X and R. ]. Clin. Microbiol. 28 (1990) 2834-2836 13. Wibawan, I. W. T. and Ch. Ldmmler: Demonstration of a and ~ components of group B streptococcal protein antigen c in serum-soft agar. J. Vet. Med. B 37 (1990) 680-683 14. Wilkinson, H. W.: Group B streptococcal infection in humans. Annu. Rev. Microbiol. 32 (1978) 41-57 Dr. Christoph Lammler, Institut fur Bakteriologie und Immunologie der ]ustus-LiebigUniversitat, Frankfurter Str. 107, D-6300 GielSen
