Vol. 1, No. 3 Printed in U.S.A.
JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1975, p. 268-273 Copyright © 1975 American Society for Microbiology
Identification of Streptococci: Serogrouping by Immunofluorescence BARBARA K. WATSON, LAWRENCE J. KUNZ,* AND ROBERT C. MOELLERING, JR. Department of Microbiology and Molecular Genetics* and Department of Medicine, Harvard Medical School, and The Francis Blake Bacteriology Laboratories and Department of Medicine Infectious Disease Unit, Massachusetts General Hospital, Boston, Massachusetts 02114 Received for publication 1 July 1974
This paper deals with the fluorescent antibody (FA) method for identifying six commonly occurring and two rare groups of streptococci by using commercially prepared (Difco) conjugates. We have shown that group-specific FA produced frequent cross-reactions with heterologous groups of organisms. These reactions varied with different strains of the same serogroup. Nonetheless, there were distinct overall patterns in the intensity and appearance of the homologous and heterologous reactions. When monitored by the precipitin test with Rantz and Randall extracts, these patterns led to the correct identification of 90 to 100% of specimens of serogroup A, B, C, and G streptococci. Many members of groups D and F also showed distinctive reaction patterns. However, there was a significant number of strains of both groups D and F that either failed to stain or stained poorly with the homologous conjugate. As a result, the identification of these serogroups by FA was less reliable. In our experience streptococci are among the groups of bacteria most frequently isolated from clinical specimens, exceeded only by the enterobacteriaceae and staphylococci. Moreover, reviews of streptococcal infections in this hospital have emphasized not only the high mortality associated with bacteremia due to various members of Streptococcaceae but also the diversity of the serogroups (including nongroupable strains) encountered in clinical specimens (5, 7). These observations prompted us to reexamine the existing methods of' classifying streptococci and to explore procedures that would permit the development of methodology for rapid and precise identif'ication of' all of the species implicated in human inf'ections. Of the serological methods, the techniques of' immunofluorescence (FA) showed suff'icient promise to warrant the commercial production of fluorescein-labeled antisera against serogroups frequently encountered clinically and industrially. This promise has not been realized because of conflicting reports of cross-reactivity of antisera and the fact that most of the interest in the use of this method has been limited to the identification of group A strains (1-3, 8, 15, 16, 20). The purpose of the experiments reported herein was to examine the effectiveness of the FA method for classifying clinical specimens of' streptococci by using commercially available conjugates.
MATERIALS AND METHODS Test strains. Except for groups E and N, the test strains consisted of clinical isolates of streptococci grown on brucella agar (Albimi Laboratories) containing 5% horse blood. Since groups E and N were not encountered in the course of the present studies, reference samples of these organisms were obtained from the Center for Disease Control, Atlanta, Ga. The growth from plate cultures was scraped off with sterile cotton swabs and suspended in 1.5-ml amounts of sterile normal saline. Portions of these suspensions were then cultured for purity, frozen (0.3 ml) with an equal volume of horse blood for future reference, inoculated (0.3 ml) into 40 ml of Todd-Hewitt broth containing 1% dextrose for use in precipitin tests, and used to make four spot smears on each of two slides. FA tests. The smears of the organisms were air dried for 15 to 30 min at 37 C, fixed in 95% ethanol for 5 to 10 min, and air dried again. The areas to be stained were scored with a diamond pencil and treated with fluorescein-labeled antisera against groups A through G and N streptococci (generously supplied by Difco Laboratories). The slides were covered with petri dishes containing moist filter paper and allowed to stand for 15 to 30 min. The conjugates were then rinsed off with a generous stream of phosphate-buffered saline (pH 7.3), and the slides were immersed in buffered saline for an additional 5 to 10 min. After gentle blotting, the spot smears were covered with 90%; glycerin in phosphate-buffered saline, a cover slip was applied, and the smears were examined under the fluorescence microscope. The optical system was a Zeiss microscope equipped with x 12.5 oculars, barrier filters that
268
269
IDENTIFYING STREPTOCOCCI BY SEROGROUPING
VOL. 1, 1975
transmitted wavelengths above 470 nm and eliminated wavelengths above 650 nm, x 10 and x40 (apochromatic, oil) objectives, a dark-field condenser, and a swing-in mirror. The light source was an Osram HBO 200 L2 high-pressure mercury vapor bulb mounted in a Zeiss fluorescence lamp assembly with a KG1 heat-absorbing filter, a BG38 red wave lengthabsorbing filter, and a UG5 fluorescence exciter filter. Because of differences in the intensity of the fluorescence produced by different conjugates, the reactions were rated as strongest, intermediate, weak, or negative in any given set of tests. Precipitin tests. The broth cultures were extracted by the autoclave method of Rantz and Randall (19) and the extracts were used in microprecipitin tests (22) with antisera to groups A through T. All grouping antisera were purchased from Burroughs, Wellcome and Co. with the exception of group D antiserum, which was kindly provided by R. Lancefield, and group T antiserum, obtained from Difco Laboratories.
of homologous and heterologous strains previously identified by the precipitin test. Preliminary attempts to titrate the conjugates indicated that, with the exception of group A antiserum, dilution resulted in a decrease in the brightness of the homologous reactions without eliminating the cross-reactions. In the case of group A antiserum, the homologous staining remained unchanged through a 1:10 dilution with only a slight decrease in the heterologous staining. Since dilute serum deteriorated rapidly on storage, all of the conjugates were used neat. The results of our tests with undiluted serum (Fig. 1) show that, although the incidence of cross-reactions varied somewhat with different strains of the same organism, there were distinct patterns in the intensity and appearance of the homologous and heterologous reactions. With the exception of group G streptococci, the homologous conjugates produced the brightest staining. The group A strains showed, in addition, weak to moderate fluorescence with antisera to groups C, D, and G but not with antisera
RESULTS The first part of the investigation was to determine the specificity of the individual conjugates. This was done by studying the staining produced in smears of a representative number
n serogrouping of streptococci by FA
Incidence anc GROUPINVG ANr/SERA
A
t
JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1975, p. 268-273 Copyright © 1975 American Society for Microbiology
Identification of Streptococci: Serogrouping by Immunofluorescence BARBARA K. WATSON, LAWRENCE J. KUNZ,* AND ROBERT C. MOELLERING, JR. Department of Microbiology and Molecular Genetics* and Department of Medicine, Harvard Medical School, and The Francis Blake Bacteriology Laboratories and Department of Medicine Infectious Disease Unit, Massachusetts General Hospital, Boston, Massachusetts 02114 Received for publication 1 July 1974
This paper deals with the fluorescent antibody (FA) method for identifying six commonly occurring and two rare groups of streptococci by using commercially prepared (Difco) conjugates. We have shown that group-specific FA produced frequent cross-reactions with heterologous groups of organisms. These reactions varied with different strains of the same serogroup. Nonetheless, there were distinct overall patterns in the intensity and appearance of the homologous and heterologous reactions. When monitored by the precipitin test with Rantz and Randall extracts, these patterns led to the correct identification of 90 to 100% of specimens of serogroup A, B, C, and G streptococci. Many members of groups D and F also showed distinctive reaction patterns. However, there was a significant number of strains of both groups D and F that either failed to stain or stained poorly with the homologous conjugate. As a result, the identification of these serogroups by FA was less reliable. In our experience streptococci are among the groups of bacteria most frequently isolated from clinical specimens, exceeded only by the enterobacteriaceae and staphylococci. Moreover, reviews of streptococcal infections in this hospital have emphasized not only the high mortality associated with bacteremia due to various members of Streptococcaceae but also the diversity of the serogroups (including nongroupable strains) encountered in clinical specimens (5, 7). These observations prompted us to reexamine the existing methods of' classifying streptococci and to explore procedures that would permit the development of methodology for rapid and precise identif'ication of' all of the species implicated in human inf'ections. Of the serological methods, the techniques of' immunofluorescence (FA) showed suff'icient promise to warrant the commercial production of fluorescein-labeled antisera against serogroups frequently encountered clinically and industrially. This promise has not been realized because of conflicting reports of cross-reactivity of antisera and the fact that most of the interest in the use of this method has been limited to the identification of group A strains (1-3, 8, 15, 16, 20). The purpose of the experiments reported herein was to examine the effectiveness of the FA method for classifying clinical specimens of' streptococci by using commercially available conjugates.
MATERIALS AND METHODS Test strains. Except for groups E and N, the test strains consisted of clinical isolates of streptococci grown on brucella agar (Albimi Laboratories) containing 5% horse blood. Since groups E and N were not encountered in the course of the present studies, reference samples of these organisms were obtained from the Center for Disease Control, Atlanta, Ga. The growth from plate cultures was scraped off with sterile cotton swabs and suspended in 1.5-ml amounts of sterile normal saline. Portions of these suspensions were then cultured for purity, frozen (0.3 ml) with an equal volume of horse blood for future reference, inoculated (0.3 ml) into 40 ml of Todd-Hewitt broth containing 1% dextrose for use in precipitin tests, and used to make four spot smears on each of two slides. FA tests. The smears of the organisms were air dried for 15 to 30 min at 37 C, fixed in 95% ethanol for 5 to 10 min, and air dried again. The areas to be stained were scored with a diamond pencil and treated with fluorescein-labeled antisera against groups A through G and N streptococci (generously supplied by Difco Laboratories). The slides were covered with petri dishes containing moist filter paper and allowed to stand for 15 to 30 min. The conjugates were then rinsed off with a generous stream of phosphate-buffered saline (pH 7.3), and the slides were immersed in buffered saline for an additional 5 to 10 min. After gentle blotting, the spot smears were covered with 90%; glycerin in phosphate-buffered saline, a cover slip was applied, and the smears were examined under the fluorescence microscope. The optical system was a Zeiss microscope equipped with x 12.5 oculars, barrier filters that
268
269
IDENTIFYING STREPTOCOCCI BY SEROGROUPING
VOL. 1, 1975
transmitted wavelengths above 470 nm and eliminated wavelengths above 650 nm, x 10 and x40 (apochromatic, oil) objectives, a dark-field condenser, and a swing-in mirror. The light source was an Osram HBO 200 L2 high-pressure mercury vapor bulb mounted in a Zeiss fluorescence lamp assembly with a KG1 heat-absorbing filter, a BG38 red wave lengthabsorbing filter, and a UG5 fluorescence exciter filter. Because of differences in the intensity of the fluorescence produced by different conjugates, the reactions were rated as strongest, intermediate, weak, or negative in any given set of tests. Precipitin tests. The broth cultures were extracted by the autoclave method of Rantz and Randall (19) and the extracts were used in microprecipitin tests (22) with antisera to groups A through T. All grouping antisera were purchased from Burroughs, Wellcome and Co. with the exception of group D antiserum, which was kindly provided by R. Lancefield, and group T antiserum, obtained from Difco Laboratories.
of homologous and heterologous strains previously identified by the precipitin test. Preliminary attempts to titrate the conjugates indicated that, with the exception of group A antiserum, dilution resulted in a decrease in the brightness of the homologous reactions without eliminating the cross-reactions. In the case of group A antiserum, the homologous staining remained unchanged through a 1:10 dilution with only a slight decrease in the heterologous staining. Since dilute serum deteriorated rapidly on storage, all of the conjugates were used neat. The results of our tests with undiluted serum (Fig. 1) show that, although the incidence of cross-reactions varied somewhat with different strains of the same organism, there were distinct patterns in the intensity and appearance of the homologous and heterologous reactions. With the exception of group G streptococci, the homologous conjugates produced the brightest staining. The group A strains showed, in addition, weak to moderate fluorescence with antisera to groups C, D, and G but not with antisera
RESULTS The first part of the investigation was to determine the specificity of the individual conjugates. This was done by studying the staining produced in smears of a representative number
n serogrouping of streptococci by FA
Incidence anc GROUPINVG ANr/SERA
A
t
