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Serological differentiation of strains of group B streptococci by the soft-agar technique
Zbl. Bakt. Hyg., I. Abt. Orig. A 249, 184-189 (1981)
Department of Microbiology and Department of Ophthalmology \ St. Marianna University School of Medicine, Sugao, Takatuku, Kawasaki 213, Japan
Serological Differentiation of Strains of Group B Streptococci
by the Soft-Agar Technique Serologische Typisierung von Stammen der Streptokokkengruppe B mit der Softagar-Technik
YOSHlTOSHl leHIMAN, ElKO KONO, HATSUYE KUSHlRO\ SEIJl ITO, and KOSAKU YOSHIDA
With 1 Figure' Received Juli 21,1980
Abstract Soft-agar technique was applied for serological determination of strains of group B streptococci. With representative type strains, their colonial morphology was converted from diffuse- to compact-type growth only by the addition of homologous rabbit antiserum in the medium and no conversion of colonial morphology was observed by heterologous rabbit antisera. Twenty seven out of 30 fresh ilolates obtained from human clinical specimens showed diffuse-type growth in soft-agar medium and were subjected to this examination. Twenty-one strains reacted with a single antiserum,S strains showed reactivities to two different antisera although reaction to an antiserum were significantly higher than those of the other antiserum and no reaction was observed with a strain. Twenty single and 5 major serotypes determined by this technique were coincided with those differentiated by Lancefield's precipitin method. From these experimental results, soft-agar technique was regarded being available for serological typing of strains of group B streptococci.
Zusammenfassung Die Softagar-Technik wurde auf ihre Anwendbarkeit zur serologischen Bestimmung von Streptokokken der Gruppe B gepriift. In Nahrboden mit Normalserum wachsen die Keime diffus, nach Zusatz typenspezifischer Antiseren erfolgt ein Umschlag des Wachstums in scharf begrenzte Kolonien. Von 30 frisch isolierten Stammen aus klinischem Material zeigten 27 in Softagar diffuses Wachstum und wurden mit Hilfe der beschriebenen Methode untersucht. Von diesen 27 Stammen reagierten 21 mit jeweils einem typenspezifischen Antiserum in Form des konkreten Koloniewachstums und konnten damit den Typen la, Ib, Ie, II bzw. III zugeordnet werden. Weitere 5 Stamrne reagierten mit 2 Antiseren. Dies wird auf das Vorkommen von zwei Antigenen zuriickgefiihrt. Ein
Serological Differentiation of Strains of Group B Streptococci
185
Stamm war nicht typisierbar. Die Ergebnisse wurden mit der konventioneIIen Prazipitationstechnik uberpriift, die zu iibereinstimmenden Ergebnisse fiihrte. Die in der SoftagarTechnik gefundenen 5 Stamme mit zwei Antigenen ergaben in der Prazipitationstechnik nur das Hauptantigen; mit der Soflagar-Technik kann die Hauptkomponente von einer Nebenantigenkomponente unterschieden werden.
Introduction
Strains of group-B streptococci possess two major polysaccharide antigens. One of them locates within cell wall distributing widely in this group of organisms as a common antigen. The other has been regarded as capsular antigen and applied for the basis subdividing group-B streptococci into 5 serotypes, la, Ib, Ie, II and III (6, 11, 12) using capillary precipitin reaction (4, 5), fluorescent antibody technique (1), and counter immunoelectrophoresis (2). On the other hand, serumsoft agar technique has been applied for the serological typing of staphylococcal capsule (13, 14). Also soft-agar technique was shown being available in the demonstration of capsular production of strains of Streptococcus pneumoniae (15), Klebsiella pneumoniae (8), Klebsiella ozaenae (9) and Escherichia coli (10). Attempts were, therefore, made whether soft-agar technique could be available in determining serotypes of group-B streptococci and interesting results were obtained. This paper is concerned with these investigations.
Materials and Methods Strains. Strains of 55-615, 55-618, 55-887, 55-619 and SS-620 were used. These were representative strains of serotypes la, Ib, Ie, II and III of group-B streptococci, respectively. These were provided from the Annex Institute, Yokosuka Navy Hospital, U. S. Navy, Sagami-Ono, Japan. Also, 30 fresh isolates of group-B streptococci, determined by the method of Lancefield (6), isolated from clinical specimens in the Clinical Laboratory, St. Marianna University Medical School Hospital, were utilized. Representative type strains and 27 fresh isolates showing diffuse-type growth in soft-agar medium determined by the method noted elsewhere (8,9,10,13,14,15), were subjected to those examination. Preparation of rabbit antisera. Formalinized organisms prepared by regular procedure were suspended in saline. On tenth ml of them were injected intravenously into rabbit weighing approximately 2 kg (Japanese white rabbit, Nihon Clea Co. Ltd, Japan) three succesive days at the first week. At second week vaccine was increased to 0.5 ml,thereafter, 1.0 ml of it was injected for one month. Two weeks after the final immunization rabbits were exsanguinated, serum was separated and was used for the experiments. Serological typing by the soft-agar technique. Organisms grown in Todd-Hewitt broth at 37 DC for overnight were diluted at one to 10-5 with sterile saline. One tenth ml of them, normal rabbit serum and rabbit antiserum were combined with 10 ml of ToddHewitt medium containing 0.1 Ofo agar (Bact agar, Difco) at the final concentration. These were kept at 4 DC cold room for 30 min to solidify the agar and were cultured at 37 DC for 18h, then, colonial morpholgy of the organisms were determined. Normal rabbit serum was used as a control for the replacement of antiserum. With this procedure, 0.1 ml of maximal antiserum dilution capable to convert to definite compact-type growth from diffuse-type growth was designated as one unit of the serum activity. For the serotyping 0.1 ml of antiserum containing two units of the activity was used. Serological typing by the precipitin test. Antigen used for serotyping was obtained by
186
Y.Iehiman, E.Kono, H.Kushiro, 5.Ito, and K. Yoshida
the method described by Pattison et a!. (7). Namely, organisms grown in Todd-Hewitt broth at 37°C for 18 h were centrifuged and the precipitate was suspended in 0.4 ml NI 5 HC!. This was heated at 50°C for two hours, the pH adjusted at 7.0 with N/2 NaOH, centrifuged and rhe supernatant was used for the antigen. Precipirin test was followed by the method of Lancefield (5) using antigen noted above.
Results
Colonial morphologies of strains of group B streptococcus in plain soft-agar medium. In plain soft-agar medium every representative serotype strains, strains 55-615, 55-618, 55-887, 55-619 and 55-620, showed diffuse-type colonial morphology. Twenty seven out of 30 (90 Ofo) fresh isolates from clinical specimens also exhibited diffuse-type growth and the remainders, 3 strains, were compact-type growth. Accordingly, compact-type strains were excluded from further experiments. Serological typing of the strains by the soft agar technique. Representative serotype strains, strains 55-615, 55-618, 55-887, 55-619 and 55-620 exhibiting diffusetype colonial morphology in plain soft-agar medium converted to compact-type growth only by the addition of homologous antisera and no change of colonial morphology was observed by the addition of heterologous antisera as shown in Fig. 1. and Table 1. When this technique was applied for 27 fresh isolates, every strain reacted with antiserum, except for one strain, exhibiting change of colonial morphologies from diffuse to compact-type growth by the addition of antiserum in soft-agar medium. With these strains 2, 2, 8, 5, and 4 strains reacted with single antiserum indicating that their types were la, Ib, Ic, II and III, respectively. With remaining 5 strains they reacted with two different antisera. Namley, in case of strain 5MU-0879, the strain showed definite compact-type colony in soft agar
Fig. 1. Colonial morphologies of strain 55-615 (Type Ia) of group B streptococci in softagar containing rabbit anti-serum prepared with representative serotype strains. A, B, C, D, E and F indicate soft-agar containing rabbit anti-serum prepared with strains 55-615 (Type Ia), 55-618 (Type rb), 55-887 (Type Ie), 55-619 (Type II), 55-620 (Type III) and normal rabbit serum, respectively.
Serological Differentiation of Strains of Group B Streptococci
187
Table 1. Colonial morphologies of representative serotype strains in soft-agar containing homologous and heterologous rabbit antiserum Ia Rabbit anti-serum Type strains C,,·,:·
I a (SS-615) I b (S5-618) I c (SS-887) II (SS-619) III (SS-620)
D D D D
Ib
Ic
II
III
NRS'"
D"""':' C D D D
D D C D D
D D D C D
D D D D C
D D D D D
':-' ".,:. and ".".". indicate normal rabbit serum, compact- and diffuse-type growth, respectively.
Table 2. Comparison of serotypes of 27 fresh isolates of group B streptococci determined by precipitin test to soft-agar technique Strain
Soft-agar technique
Precipitin test
Strain
Soft-agar technique
Precipitin test
SMU-0009 SMU-0880 SMU-0879 SMU-1903 SMU-0012 SMU-1782 SMU-5302 SMU-1280 SMU-2439 SMU-631O SMU-0007 SMU-4976 SMU-1425 SMU-2118 SMU-4715 SMU-0918
Ia Ia I a, Ib Ib Ib Ic Ic Ic Ic Ic Ic Ic Ic I c, I b I c, I b I c, I b
Ia Ia Ia Ib Ib Ic Ic Ic Ic Ic Ic Ic Ic Ic Ic Ic
SMU-3111 SMU-5902 SMU-1381 SMU-0422 SMU-3832 SMU-0008 SMU-0001 SMU-1352 SMU-0891 SMU-1452 SMU-1782
II II II II II III III III
II II II II II III III III III
III I b, I c
NT
NT NT
medium containing type Ia antiserum, however, short tailing appeared with compact-type colonies in the medium containing type Ib antiserum. These findings indicate that major type antigen of this strain was Ia and Ib antigen also existed as a minor component. Similarly, with the strains of SMU-2118, -4715 and -0918, they produced definite compact-type colony against type Ie antiserum and short tailing was appeared against type Ib antiserum. Also, strain SMU-1452 showed definite compact-type against type Ib antiserum and produced compact-type colony forming short tail by type Ie antiserum. Precipitin test of strains of group-B streptococcus. To compare the reactions of standard type strains in soft-agar containing antisera, they were reexamined by Lancefield's precipitin method (4, 5). Experimental results showed that Hel ex-
188
Y.Ichiman, E.Kana, H. Kushiro, S.Ito, and K.Yoshida
tract of the strain 55-615, type la, reacted with antiserum to the strains, type Ia and Ie. With the HCl extract of the strains 55-618, type Ib, reacted both antiserum to the strains type Ib and Ie. In case of the extract of the strain 55-887, type Ie reacted with antisera to the strains homologous, 55-615 and 55-618 and the extracts from 55-619, type II, and 55-620, type III, reacted only homologous antisera. Subsequently, 27 fresh isolates were subjected to the precipitin test. Then, every strain including major type of 5 strains exhibiting bivalent serotype by the softagar technique, coincided with the experimental results obtained by the precipitin test. However, serotype patterns of these bivalent strains by soft-agar technique were quite agreeable by the reexamination of precipitin test noted above. Two of the three strains, showing compact type growth in soft-agar containing antisera were non-typable by the precipitin test and the third was identified as type Ia.
Discussion Type specific polysaccharide antigens of strains of group B streptococci have been regarded not to be native form in hot-HCl extracts (4, 5, 7). Although sufficient results were obtained in differentiation of the strains for epidemiological purpose, serological determination of group B streptococci is assumed being based on the denatured antigens. On the other hand, relative cell surface structure of several species of bacteria (8, 9, 10, 13, 14, 15) relates to the colonial morphologies of these organisms in soft-agar or serum-soft agar media. It would be possible, therefore, that conversion of colonial morphology by the addition of corresponding antisera is due to the results of serological reactions between specific antibodies and antigens in most outer layer of the organisms multiplying in soft-agar media. In these experiments, 27 (90 %) out of 30 fresh isolates exhibiting diffuse-type growth in soft-agar medium were subjected to these examinations. Except a strain, for every strain was differentiated the serological type by this technique. With these fresh isolates 21 strains reacted with single type antiserum and the others reacted with two different type antisera. However, it was possible to determine which type antigen was major or minor for the strains by reading the colonial morphologies converted by the addition of specific antisera. These findings coincided with experimental results achieved by the method of Lancefield (5). From these results it was assumed that soft agar technique was available for the serological typing of group B streptococci. In this case, a small amount of anti-type serum was enough for serological typing and the result obtained was higher in sensitivity comparing to that of the precipitin test (5, 7). Also, extraction of the antigens is not required for performing this technique as in capillary precipitin test (7), fluorescein antibody technique (1) and immunoelectrophoresis (2). However, with this technique strains showing compact-type growth in plain soft-agar are not available submitting to the examination at present time. In case of staphylococcal capsular type antigen, Yoshida et a1. (13, 14) detected it in unencapsulated organisms, compact-type in regular serum-soft agar medium. Accordingly compact-type strains of group B streptococci would be possible to possess type antigents) although Kane et a1. (3) noted that compact-type strains of group B streptococci were unencapsulated. These problems are currently under investigations in our laboratory.
Serological Differentiation of Strains of Group B Streptococc i
189
References 1. Cropp, C. T., R. A. Zimmerman, ]. [elinhoua, A. H. Avernhaimer, R. A. Bolin, and B. C. Wyric: Sero typing of group-B streptococci by slide agglutination , fluorescent micros copy and microimmunodiffusion. J. Lab. Clin . Med. 84 (1974) 594-603 Z. Hill, H. R.. M. f. Riter. S. K. Menge. D. R. Johnson, and j. M. Ml1tMJI! R:\~id identification of group B strepto cocci by counter imunoelectropho resis. J. Clin. Microbiol. 1 (1975) 188-191
3. Kane, ]. A., A. E. Rabkin, and W. W. Karakau/a. Dem onst ration of the capsular antigens of bovine group B streptococci by the serum-soft agar meth od. ]. Clin. MicrobioI. 2 (1975) 35-41 4. Lancefield, R. C.: A serological different iation of specific types of bovine hemolytic streptococci (group B).]. expo Med. 59 (1934) 441-458 5. Lancefield, R. C.: T wo serological types of group B hemolytic streptococci with related but not identical type-specific substances. ]. expo Med. 67 (1938) 25-40 6. Lancefield, R. C., M. McCarty, and W. N. Everly: Mul tiple mous e protective antibodies directed aga inst gro up B streptococci. J. exp o Med. 142 (1975) 165-179 7. Pattison, I. H., P. R. Matthews, and D. G. Howell: T ype classification by Lancefield's precip itin meth od of hu man and bovine group B stre pto cocci isolated in Britain. ]. Path. Bacr. 69 (1955) 43-50 8. Takahashi, M., K. Yoshida, and C. L. San Clemente: Relation of colonial morphologies in soft agar to morpholog ical and biological prop ert ies of the K-9 strain of Klebsiella pneumoniae and its va riants. Canad. J. M icrobiol. 23 (1977) 448-4 51 9. Takahashi, M., S. lwami, and K. Yoshida: Mouse viru lence of a strain of Klebsiella ozaenea and its variant s an relati on to colonial morphologies in soft agar. Microbiol. Immu nol. 22 (1978) 361- 363 10.Takahashi, M., Y.lchiman, Y. Minegishi, and K. Yoshida: Relation of colonial morph olog ies of strains of Escherichia coli in soft-aga r to morphological and biological prop ert ies. l ap. ] . Bact. 34 (1979) 248 11.Wilkinson, H . W. and R. G. Eagan: T ype specific antigens of group B type Ie strepto cocci. Infect, Immun . 4 (1971) 596-604 12.Wilkinson, H . W.: Immunochemistry of purified polysaccharide type antigens of group B streptococc al types la, Ib and Ie. Infect. Immun. 11 (1975) 845- 852 13. Yoshida, K.: Dem onstrati on of sero logically different capsular types amo ng strains of Staphylococcus aureus by the seru m-soft aga r technique. Infect. Jmmun . 3 (1971) 535539 14. Yoshida, K.: Isolation of an additional capsular type strains of Staphylococcus aureus by the serum-soft agar technique. Infect. Immun. 5 (1972) 833- 834 15.Yoshida, K., M. Takahashi, Y.lchiman, S. Narikawa, and E. Kana: Relati on of colonial morphologies of str ain of Streptococcus pneumoniae in soft-agar to the encapsulat ion . Asian ] . Infect. Dis. (1980) (in pr ess) Dr. Yi lchiman, Dept. of Mic robiology, St. M arianna University School of Medi cine, Sugao, T akatu-ku, Kawasaki 213, Japan
Department of Microbiology and Department of Ophthalmology \ St. Marianna University School of Medicine, Sugao, Takatuku, Kawasaki 213, Japan
Serological Differentiation of Strains of Group B Streptococci
by the Soft-Agar Technique Serologische Typisierung von Stammen der Streptokokkengruppe B mit der Softagar-Technik
YOSHlTOSHl leHIMAN, ElKO KONO, HATSUYE KUSHlRO\ SEIJl ITO, and KOSAKU YOSHIDA
With 1 Figure' Received Juli 21,1980
Abstract Soft-agar technique was applied for serological determination of strains of group B streptococci. With representative type strains, their colonial morphology was converted from diffuse- to compact-type growth only by the addition of homologous rabbit antiserum in the medium and no conversion of colonial morphology was observed by heterologous rabbit antisera. Twenty seven out of 30 fresh ilolates obtained from human clinical specimens showed diffuse-type growth in soft-agar medium and were subjected to this examination. Twenty-one strains reacted with a single antiserum,S strains showed reactivities to two different antisera although reaction to an antiserum were significantly higher than those of the other antiserum and no reaction was observed with a strain. Twenty single and 5 major serotypes determined by this technique were coincided with those differentiated by Lancefield's precipitin method. From these experimental results, soft-agar technique was regarded being available for serological typing of strains of group B streptococci.
Zusammenfassung Die Softagar-Technik wurde auf ihre Anwendbarkeit zur serologischen Bestimmung von Streptokokken der Gruppe B gepriift. In Nahrboden mit Normalserum wachsen die Keime diffus, nach Zusatz typenspezifischer Antiseren erfolgt ein Umschlag des Wachstums in scharf begrenzte Kolonien. Von 30 frisch isolierten Stammen aus klinischem Material zeigten 27 in Softagar diffuses Wachstum und wurden mit Hilfe der beschriebenen Methode untersucht. Von diesen 27 Stammen reagierten 21 mit jeweils einem typenspezifischen Antiserum in Form des konkreten Koloniewachstums und konnten damit den Typen la, Ib, Ie, II bzw. III zugeordnet werden. Weitere 5 Stamrne reagierten mit 2 Antiseren. Dies wird auf das Vorkommen von zwei Antigenen zuriickgefiihrt. Ein
Serological Differentiation of Strains of Group B Streptococci
185
Stamm war nicht typisierbar. Die Ergebnisse wurden mit der konventioneIIen Prazipitationstechnik uberpriift, die zu iibereinstimmenden Ergebnisse fiihrte. Die in der SoftagarTechnik gefundenen 5 Stamme mit zwei Antigenen ergaben in der Prazipitationstechnik nur das Hauptantigen; mit der Soflagar-Technik kann die Hauptkomponente von einer Nebenantigenkomponente unterschieden werden.
Introduction
Strains of group-B streptococci possess two major polysaccharide antigens. One of them locates within cell wall distributing widely in this group of organisms as a common antigen. The other has been regarded as capsular antigen and applied for the basis subdividing group-B streptococci into 5 serotypes, la, Ib, Ie, II and III (6, 11, 12) using capillary precipitin reaction (4, 5), fluorescent antibody technique (1), and counter immunoelectrophoresis (2). On the other hand, serumsoft agar technique has been applied for the serological typing of staphylococcal capsule (13, 14). Also soft-agar technique was shown being available in the demonstration of capsular production of strains of Streptococcus pneumoniae (15), Klebsiella pneumoniae (8), Klebsiella ozaenae (9) and Escherichia coli (10). Attempts were, therefore, made whether soft-agar technique could be available in determining serotypes of group-B streptococci and interesting results were obtained. This paper is concerned with these investigations.
Materials and Methods Strains. Strains of 55-615, 55-618, 55-887, 55-619 and SS-620 were used. These were representative strains of serotypes la, Ib, Ie, II and III of group-B streptococci, respectively. These were provided from the Annex Institute, Yokosuka Navy Hospital, U. S. Navy, Sagami-Ono, Japan. Also, 30 fresh isolates of group-B streptococci, determined by the method of Lancefield (6), isolated from clinical specimens in the Clinical Laboratory, St. Marianna University Medical School Hospital, were utilized. Representative type strains and 27 fresh isolates showing diffuse-type growth in soft-agar medium determined by the method noted elsewhere (8,9,10,13,14,15), were subjected to those examination. Preparation of rabbit antisera. Formalinized organisms prepared by regular procedure were suspended in saline. On tenth ml of them were injected intravenously into rabbit weighing approximately 2 kg (Japanese white rabbit, Nihon Clea Co. Ltd, Japan) three succesive days at the first week. At second week vaccine was increased to 0.5 ml,thereafter, 1.0 ml of it was injected for one month. Two weeks after the final immunization rabbits were exsanguinated, serum was separated and was used for the experiments. Serological typing by the soft-agar technique. Organisms grown in Todd-Hewitt broth at 37 DC for overnight were diluted at one to 10-5 with sterile saline. One tenth ml of them, normal rabbit serum and rabbit antiserum were combined with 10 ml of ToddHewitt medium containing 0.1 Ofo agar (Bact agar, Difco) at the final concentration. These were kept at 4 DC cold room for 30 min to solidify the agar and were cultured at 37 DC for 18h, then, colonial morpholgy of the organisms were determined. Normal rabbit serum was used as a control for the replacement of antiserum. With this procedure, 0.1 ml of maximal antiserum dilution capable to convert to definite compact-type growth from diffuse-type growth was designated as one unit of the serum activity. For the serotyping 0.1 ml of antiserum containing two units of the activity was used. Serological typing by the precipitin test. Antigen used for serotyping was obtained by
186
Y.Iehiman, E.Kono, H.Kushiro, 5.Ito, and K. Yoshida
the method described by Pattison et a!. (7). Namely, organisms grown in Todd-Hewitt broth at 37°C for 18 h were centrifuged and the precipitate was suspended in 0.4 ml NI 5 HC!. This was heated at 50°C for two hours, the pH adjusted at 7.0 with N/2 NaOH, centrifuged and rhe supernatant was used for the antigen. Precipirin test was followed by the method of Lancefield (5) using antigen noted above.
Results
Colonial morphologies of strains of group B streptococcus in plain soft-agar medium. In plain soft-agar medium every representative serotype strains, strains 55-615, 55-618, 55-887, 55-619 and 55-620, showed diffuse-type colonial morphology. Twenty seven out of 30 (90 Ofo) fresh isolates from clinical specimens also exhibited diffuse-type growth and the remainders, 3 strains, were compact-type growth. Accordingly, compact-type strains were excluded from further experiments. Serological typing of the strains by the soft agar technique. Representative serotype strains, strains 55-615, 55-618, 55-887, 55-619 and 55-620 exhibiting diffusetype colonial morphology in plain soft-agar medium converted to compact-type growth only by the addition of homologous antisera and no change of colonial morphology was observed by the addition of heterologous antisera as shown in Fig. 1. and Table 1. When this technique was applied for 27 fresh isolates, every strain reacted with antiserum, except for one strain, exhibiting change of colonial morphologies from diffuse to compact-type growth by the addition of antiserum in soft-agar medium. With these strains 2, 2, 8, 5, and 4 strains reacted with single antiserum indicating that their types were la, Ib, Ic, II and III, respectively. With remaining 5 strains they reacted with two different antisera. Namley, in case of strain 5MU-0879, the strain showed definite compact-type colony in soft agar
Fig. 1. Colonial morphologies of strain 55-615 (Type Ia) of group B streptococci in softagar containing rabbit anti-serum prepared with representative serotype strains. A, B, C, D, E and F indicate soft-agar containing rabbit anti-serum prepared with strains 55-615 (Type Ia), 55-618 (Type rb), 55-887 (Type Ie), 55-619 (Type II), 55-620 (Type III) and normal rabbit serum, respectively.
Serological Differentiation of Strains of Group B Streptococci
187
Table 1. Colonial morphologies of representative serotype strains in soft-agar containing homologous and heterologous rabbit antiserum Ia Rabbit anti-serum Type strains C,,·,:·
I a (SS-615) I b (S5-618) I c (SS-887) II (SS-619) III (SS-620)
D D D D
Ib
Ic
II
III
NRS'"
D"""':' C D D D
D D C D D
D D D C D
D D D D C
D D D D D
':-' ".,:. and ".".". indicate normal rabbit serum, compact- and diffuse-type growth, respectively.
Table 2. Comparison of serotypes of 27 fresh isolates of group B streptococci determined by precipitin test to soft-agar technique Strain
Soft-agar technique
Precipitin test
Strain
Soft-agar technique
Precipitin test
SMU-0009 SMU-0880 SMU-0879 SMU-1903 SMU-0012 SMU-1782 SMU-5302 SMU-1280 SMU-2439 SMU-631O SMU-0007 SMU-4976 SMU-1425 SMU-2118 SMU-4715 SMU-0918
Ia Ia I a, Ib Ib Ib Ic Ic Ic Ic Ic Ic Ic Ic I c, I b I c, I b I c, I b
Ia Ia Ia Ib Ib Ic Ic Ic Ic Ic Ic Ic Ic Ic Ic Ic
SMU-3111 SMU-5902 SMU-1381 SMU-0422 SMU-3832 SMU-0008 SMU-0001 SMU-1352 SMU-0891 SMU-1452 SMU-1782
II II II II II III III III
II II II II II III III III III
III I b, I c
NT
NT NT
medium containing type Ia antiserum, however, short tailing appeared with compact-type colonies in the medium containing type Ib antiserum. These findings indicate that major type antigen of this strain was Ia and Ib antigen also existed as a minor component. Similarly, with the strains of SMU-2118, -4715 and -0918, they produced definite compact-type colony against type Ie antiserum and short tailing was appeared against type Ib antiserum. Also, strain SMU-1452 showed definite compact-type against type Ib antiserum and produced compact-type colony forming short tail by type Ie antiserum. Precipitin test of strains of group-B streptococcus. To compare the reactions of standard type strains in soft-agar containing antisera, they were reexamined by Lancefield's precipitin method (4, 5). Experimental results showed that Hel ex-
188
Y.Ichiman, E.Kana, H. Kushiro, S.Ito, and K.Yoshida
tract of the strain 55-615, type la, reacted with antiserum to the strains, type Ia and Ie. With the HCl extract of the strains 55-618, type Ib, reacted both antiserum to the strains type Ib and Ie. In case of the extract of the strain 55-887, type Ie reacted with antisera to the strains homologous, 55-615 and 55-618 and the extracts from 55-619, type II, and 55-620, type III, reacted only homologous antisera. Subsequently, 27 fresh isolates were subjected to the precipitin test. Then, every strain including major type of 5 strains exhibiting bivalent serotype by the softagar technique, coincided with the experimental results obtained by the precipitin test. However, serotype patterns of these bivalent strains by soft-agar technique were quite agreeable by the reexamination of precipitin test noted above. Two of the three strains, showing compact type growth in soft-agar containing antisera were non-typable by the precipitin test and the third was identified as type Ia.
Discussion Type specific polysaccharide antigens of strains of group B streptococci have been regarded not to be native form in hot-HCl extracts (4, 5, 7). Although sufficient results were obtained in differentiation of the strains for epidemiological purpose, serological determination of group B streptococci is assumed being based on the denatured antigens. On the other hand, relative cell surface structure of several species of bacteria (8, 9, 10, 13, 14, 15) relates to the colonial morphologies of these organisms in soft-agar or serum-soft agar media. It would be possible, therefore, that conversion of colonial morphology by the addition of corresponding antisera is due to the results of serological reactions between specific antibodies and antigens in most outer layer of the organisms multiplying in soft-agar media. In these experiments, 27 (90 %) out of 30 fresh isolates exhibiting diffuse-type growth in soft-agar medium were subjected to these examinations. Except a strain, for every strain was differentiated the serological type by this technique. With these fresh isolates 21 strains reacted with single type antiserum and the others reacted with two different type antisera. However, it was possible to determine which type antigen was major or minor for the strains by reading the colonial morphologies converted by the addition of specific antisera. These findings coincided with experimental results achieved by the method of Lancefield (5). From these results it was assumed that soft agar technique was available for the serological typing of group B streptococci. In this case, a small amount of anti-type serum was enough for serological typing and the result obtained was higher in sensitivity comparing to that of the precipitin test (5, 7). Also, extraction of the antigens is not required for performing this technique as in capillary precipitin test (7), fluorescein antibody technique (1) and immunoelectrophoresis (2). However, with this technique strains showing compact-type growth in plain soft-agar are not available submitting to the examination at present time. In case of staphylococcal capsular type antigen, Yoshida et a1. (13, 14) detected it in unencapsulated organisms, compact-type in regular serum-soft agar medium. Accordingly compact-type strains of group B streptococci would be possible to possess type antigents) although Kane et a1. (3) noted that compact-type strains of group B streptococci were unencapsulated. These problems are currently under investigations in our laboratory.
Serological Differentiation of Strains of Group B Streptococc i
189
References 1. Cropp, C. T., R. A. Zimmerman, ]. [elinhoua, A. H. Avernhaimer, R. A. Bolin, and B. C. Wyric: Sero typing of group-B streptococci by slide agglutination , fluorescent micros copy and microimmunodiffusion. J. Lab. Clin . Med. 84 (1974) 594-603 Z. Hill, H. R.. M. f. Riter. S. K. Menge. D. R. Johnson, and j. M. Ml1tMJI! R:\~id identification of group B strepto cocci by counter imunoelectropho resis. J. Clin. Microbiol. 1 (1975) 188-191
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