Pennsylvania Md.) to obtain. coagglutination. incubated at 35 C under anaerobic conditions in type

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1980, p /80/ /05$02.00/0 Vol. 12, No. 4 Serogrouping Single Colonies of Beta-Hemolytic Streptococci from Primary Throat Culture Plates with Nitrous Acid Extraction and Phadebact Streptococcal Reagents MALCOLM SLIFKIN* AND GAIL INTERVAL Section of Microbiology, Department of Laboratory Medicine, Allegheny General Hospital, Pittsburgh, Pennsylvania The serogrouping of isolated beta-hemolytic streptococcal colonies from throat cultures by a micronitrous acid extraction method employing Phadebact Streptococcus Test reagents was compared with results obtained with the direct-plate Phadebact procedure and the autoclave and Streptomyces albus enzyme-lysozyme extraction methods. These data were compared with those from the Lancefield grouping obtained with a capillary precipitin test. The micronitrous acid extraction method was modified to yield an uncomplicated method that provides specific coagglutination responses from one beta-hemolytic streptococcal colony that may be collected from a primary blood agar plate either on the end of an applicator stick or from a sweep of an inoculating loop from mixed growth. We have previously demonstrated that the containing 15% (vol/vol) glycerol at -70 C (14). Clinical specimens received on transport swabs (Cultu- Phadebact Streptococcus Test can be employed with the use of a minimum of five well-isolated rette; Marion Scientific Corp., Rockford, Ill.) and the beta-hemolytic streptococcal colonies derived stock cultures were streaked on Columbia sheep blood from a primary blood agar isolation plate from agar plates (BBL Microbiology Systems, Cockeysville, throat cultures (14, 15). Recently, the Md.) to obtain coagglutination procedure was modified so as to permit A Bio-Bags with catalast and anaerobic generator isolated organisms. The plates were incubated at 35 C under anaerobic conditions in type the serogrouping of five well-isolated colonies (Marion Scientific Corp.) for 12 to 18 h. The plates from primary plates that were extracted with a were then observed for the presence of beta-hemolytic mixture of Streptomyces albus enzyme and lysozyme (2). and the determination of hemolytic and catalase activ- streptococci with the application of Gram straining The nitrous acid extraction (16) was previously described as a method that can extract the Isolated colonies of beta-hemolytic streptococci ity (5, 8). group-specific carbohydrate from streptococcal from each clinical primary plate and stock cultures cell walls from single colonies as well as from were selected and employed for the direct-plate coagglutination method with the Phadebact methods (14). tonsillar scrapings (3). The nitrous extraction Other isolated colonies of beta-hemolytic streptococci method was also shown to extract more group were respectively extracted for their group-specific antigen than either the Lancefield hot HC1 or antigen by means of the autoclave, enzyme, or nitrous Fuller formamide techniques (3). acid extraction procedures. Similarly, other bacterial We describe the application of a micronitrous species were extracted by the latter procedures and acid extraction method in combination with the examined for their cross-reactivities with the Phadebact reagents. In some experiments, single colonies Phadebact Streptococcus Test reagents for the seroidentification of single colonies of beta-hemolytic streptococci obtained froui mixed cul- applicator stick to a colony and transferring it to a 0.5- were obtained by touching the end of a sterile wooden tures from primary throat dram culture plates. The (ca. 0.8-g), screw-capped specimen bottle (Fisher Scientific) containing one of the three extraction nitrous extraction method will be compared with fluids; in other experiments, extractions were obtained the autoclave method (6, 7, 13) and the S. albuslysozyme extraction methods (18) as well as the a primary culture plate so as to collect a single colony by means of an inoculating loop that was swept across direct (five-colony) method with the Phadebact of beta-hemolytic streptococci as well as surrounding reagents. background bacterial flora. In other instances, single well-isolated colonies were inoculated into 40 ml of Todd-Hewitt broth MATERIALS (BBL) for use in the Lancefield AND METHODS grouping by precipitin testing as a reference method. Bacterial strains tested. The cultures of bacteria A total of 171 primary isolates of beta-streptococci were obtained from clinical specimens and stock cultures. Stock cultures of sero beta-hemolytic primary isolates were sero with the direct- were derived from throat cultures. These 12- to 18-h streptococci were maintained in Trypticase soy broth plate Phadebact method when possible and from ex- 541

2 542 SLIFKIN AND INTERVAL tracts of single beta-hemolytic streptococcal colonies obtained from the three extraction methods. The results were compared with those obtained by the capillary precipitin method. Reagents. The Phadebact Streptococcus Test reagents (Pharmacia Diagnostics) containing lyophilized group A, B, C, and G reagents were reconstituted as suggested by the manufacturer. Coagglutination procedures. (i) Direct-plate method. The direct-plate method is described elsewhere (14). Briefly, five well-isolated beta-hemolytic colonies were picked from the primary culture plates with an applicator stick and mixed with a drop of Phadebact buffer on a microscope slide. A drop of Phadebact reagent was then added and mixed. The slide was then rocked for 1 min and examined for coagglutination with transillumination against a dark background. (ii) Modified autoclave extraction method (6, 7). One or more colonies of beta-hemolytic streptococci or other bacterial species were added to 0.2 ml of 0.9% saline in a 0.5-dram screw-capped specimen bottle (Fisher). A nonabsorbent cotton plug was placed in the open end of the tube, and the mixture was autoclaved at 121 C for 15 min. After centrifugation the supernatant fluid was employed to serogroup one or more colonies with the Phadebact reagents as described above. (iii) S. albus-lysozyme enzyme extraction method (2). One or more beta-streptococcal colonies or other bacterial species were suspended in 0.2 ml of S. albus enzyme-lysozyme extraction solution in a 0.5- dram (ca. 8.0-g) screw-capped specimen bottle. The extraction solution was prepared by reconstituting S. albus filtrate (Difco) with an aqueous solution of 5 mg of lysozyme per ml (18) (Sigma Chemical Co., St. Louis, Mo.; Difco). The cell suspension was incubated in the extraction mixture at 55 C for 30 min and centrifuged at 1,000 x g for 30 min. Separate drops of the extract were tested with the respective Phadebact reagents. (iv) Micronitrous acid extraction method (3). One or more colonies of beta-hemolytic streptococci or other bacterial species were placed in a 0.5-dram screw-capped specimen bottle containing 20,ul of a 2 M sodium nitrite solution. A 3-1l amount of glacial acetic acid was then added. After an incubation period of 15 min at room temperature, a small amount (16 to 24 mg) of sodium bicarbonate was placed on a microspatula and added to the mixture. The solution was brought to a final volume of 80,ul with distilled water. Individual drops of the extract uncentrifuged were respectively mixed with 1 drop of individual Phadebact reagents and examined for coagglutination response. (v) Lancefield grouping. Extracts were prepared for the Lancefield grouping (10) by the autoclave extraction method (13), using 18-h cultures in 40 ml of Todd-Hewitt broth. Commercial antisera (Burroughs Wellcome) for groups A, B, C, D, F, and G were employed in the performance of the precipitin test in capillary tubes (17). J. CLIN. MICROBIOL. RESULTS Sensitivity. The minimal number of betahemolytic streptococci colonies yielding detectable specific-group polysaccharide from 15 strains each of group A, B, C, and G streptococci, as determined with Phadebact coagglutination reagents, generally varied from one to five colonies with the autoclave and enzyme extraction methods. In contrast, the nitrous acid method required only a single colony for the extraction of specific serogroupable substance with all the beta-streptococcal groups examined (Table 1). The variation in minimal number of colonies required for the specific coagglutination response with the autoclave and enzyme extraction methods appeared to be associated with relative size of the colonies selected. Accordingly, some strains of the respective streptococci tested were somewhat larger than others. Furthermore, those colonies selected from isolated areas of the blood agar plate were relatively larger than those closely adjacent to other colonies. However, the relatively small colonies, particularly those representing group C, were always serogroupable by coagglutination by micronitrous acid extractions of their single colonies. Cross-reactivity. Cross-reactive coagglutination response occurred when autoclave and enzyme extracts derived from each of one colony obtained from 50 strains of Streptococcus pneumoniae were mixed with Phadebact group C reagent. The coagglutination was manifested after 40 to 80 s of mixing the reactants on microscope slides. However, no serological cross-reactions occurred with single colonies of this bacterium when the group C reagent and the nitrous acid extracts were employed. Cross-reactivity for group C Phadebact reagents was not observed with the other bacterial species examined with any of the three extraction methods (Table 2). No cross-reactions occurred between any of the non-streptococcal strains examined and the Phadebact reagents for groups A, B, and G A determination of the effectiveness of the TABLE 1. Comparison of minimal number of betahemolytic streptococci required for serogrouping with Phàdebact reagents by three extraction methods Minimal no. of colonies yielding coagglutination for sero- Extraction method group: A B C G Autoclave S. albus enzyme-lysozyme Nitrous acid

3 VOL. 12, 1980 TABLE 2. Comparison of cross-reactivity of various bacteria extracted by three methods with group C Phadebact reagent Extraction method Organism No. examined Auto- Enzymeb Nitrous clave' acid' S. pneumo- 50 5od 50 0 niae All others a Autoclave-extraction in 0.2 ml of saline. b S. albus-lysozyme extraction in 0.2 ml of enzyme solution, 55 C, 30 min. c Micronitrous acid extraction. d Number of isolates reacting with group C reagents. e Other bacteria tested: Citrobacter diversus (6), Enterobacter cloacae (6), Escherichia coli (6), Pseudomonas aeruginosa (5), Serratia liquefaciens (6), Viridans streptococci (10), and Staphylococcus aureus (5). nitrous acid extraction method for the serogrouping of beta-hemolytic streptococci from mixed growth was next pursued. Sweeps with a loop were obtained from 10 primary plates containing 4 to 300 beta-hemolytic colonies. Each sweep was prepared so as to collect one betahemolytic colony along with a mixture of other throat flora adjacent to that colony. The inocula were then respectively extracted with nitrous acid and examined for specific serogrouping responses as well as for cross-reactive responses with the Phadebact reagents. The coagglutination reaction occurring with the mixed culture method from all the 10 primary culture plates yielded the correct serogrouping with the nitrous acid extract as compared with the results of Lancefield grouping performed on pure cultures of the respective beta-hemolytic streptococci by the autoclave method of extraction. Furthermore, cross-reactions were not obtained with any of the Phadebact reagents when the mixed culture procedures were employed. The Phadebact test was performed on 171 primary clinical isolates derived from 171 respiratory specimens using the micronitrous acid extraction method. For this investigation, single beta-hemolytic streptococci colonies were respectively transferred on the end ofan applicator stick. No care was taken to pick colonies that were completely isolated from background flora. This procedure, when compared with the precipitin method, permitted the correct identification of all the 171 isolates examined. The direct (fivecolony) Phadebact test permitted the correct identification of the 68 primary beta-hemolytic streptococci isolates which could be tested by this method. These results are presented in Table 3. MICRONITROUS ACID EXTRACTION AND PHADEBACT 543 TABLE 3. Comparison between the single-colony nitrous acid extraction methods and the direct method for serogrouping beta-hemolytic streptococci from primary isolation plates employing Phadebact coagglutination reagents Direct methoda Nitrous traction acid ex- Rantz-Ran- dal autoclave ex- Group traction No. No. cor- No. No. cor- (No. tested rectly rectly Lancefield by procedure) A B C G Non a Minimal number of 5 well-isolated colonies employed. DISCUSSION The nitrous acid extraction method was previously demonstrated to be a specific and practical means to serogroup beta-hemolytic streptococci by the capillary precipitin technique from the cells of overnight broth cultures (4) as well as from the growth of 18-h cultures from one-half sheep blood agar plates (4). The present investigation has shown the micronitrous acid extraction method to be applicable to the coagglutination procedure with Phadebact reagents. One major modification of this procedure is the addition of a relatively smail amount of sodium bicarbonate to neutralize the reaction mixture in place of the previously described method which employed a ph indicator and 0.5 (4) or 10 N NaOH (3) to adjust the reaction mixture to ph 7.5. The use of the bicarbonate for the neutralization step is a rather uncomplicated procedure that is performed in a single step in contrast to the latter method. Furthermore, the use of the bicarbonate changed the ph values of the reaction mixture in a range of 6.8 to 7.9. These ph values permitted the specific coagglutination responses to occur optimally. Unlike the micronitrous extraction methods previously described (3), the reaction mixture was brought to a final volume of 80,ul instead of 50,ul. This increase in volume was necessary for respectively testing an extract with each of the four Phadebact reagents and did not appear to decrease the accuracy of the coagglutination responses. The S. albus enzyme-lysozyme extraction method has previously been employed as the means to prepare extracts of five well-isolated beta-streptococci colonies with laboratory-prepared coagglutination reagents (2). In that report, five colonies were chosen as the minimum

4 544 SLIFKIN AND INTERVAL number since less than that number of colonies did not yield enough extractable polysaccharide group antigen for a serological reaction to occur with all the serogroups examined. In this investigation it was demonstrated that the autoclave extraction method as well as the enzyme extraction method also would require approximately five colonies to yield extractable group antigen for each of the serogroups examined. In another study employing S. albus enzyme, however, without lysozyme, Matthieu et al. observed that two or three colonies of group A beta-hemolytic streptococci were required to yield an easily interpretable coagglutination response (12). These investigators observed that with minute beta-hemolytic streptococcal colonies more colonies were required to obtain a serological reaction. Furthermore, they pointed out that well-isolated beta-hemolytic streptococci colonies are required for the S. albus enzyme treatment. In the present investigation it was shown that the micronitrous extraction required only a single colony to correctly seroidentify all the beta-streptococcal isolates derived from primary culture plates. Furthermore, a second significant observation concerning the micronitrous extraction method should be stressed. There is no need to employ well-isolated betahemolytic streptococcal colonies with the micronitrous acid extraction method. As shown in this investigation, the micronitrous acid extraction method, as opposed to the enzyme and autoclave extraction methods, does not yield cross-reactive antigenic substances from S. pneumoniae that seroreact with group C antibodies (1, 9, 11, 15). Thus, this investigation confirms the sensitivity of the micronitrous extraction method (3) and demonstrates that only specific group antigens are detected by this extraction procedure with the Phadebact reagents. Therefore, a mixed-growth procedure, using the micronitrous extraction method with Phadebact reagents, appears to be devoid of the cross-reactivity problems with background flora that are associated with the mixed-growth procedure of the Streptex Latex Agglutination Test Kit (15), which utilizes pronase as the extraction enzyme. Although a Phadebact reagent for group D streptococci was not available during this investigation, this coagglutination reagent is presently commercially available. However, the group A, B, C, and G Phadebact reagents will suffice for serogrouping beta-hemolytic streptococci from throat specimens. Group D streptococci are not usually associated with throat specimens, and if they are, they are of little clinical significance (6) Ȧ potential experimental bias due to variation J. CLIN. MICROBIOL. between lots of coagglutination reagent was apparently eliminated since similar results of sensitivity and specificity occurred with the three different lots of Phadebact reagents employed in this investigation. Furthermore, the grouping methods were performed blind, relative to each other. In summary, the use of the micronitrous acid extraction method and Phadebact reagents with primary beta-hemolytic streptococcal colonies derived from throat cultures provides a relatively uncomplicated and sensitive procedure for the accurate serogrouping of beta-hemolytic streptococci. The procedure allows rapid group identification using one streptococcal colony with Phadebact Streptococcus Test reagents and may be employed with mixed growth without cross-reactivity of group C antibody. LITERATURE CITED 1. Austrian, R., C. Buettger, and M. Dale Problems in the classification and pathogenic role of alpha and nonhemolytic streptococci of the human respiratory tract, p In L. W. Wannamaker and J. M. Matsen (ed.), Streptococci and streptococcal diseases. Academic Press Inc., New York. 2. Carlson, J. R., and L. R. McCarthy Modified coagglutination procedure for the serological grouping of streptococci. J. Clin. Microbiol. 9: El Kholy, A., R. Facklam, G. Sabre, and J. Rotta Serological identification of group A streptococci from throat scrapings before culture. J. Clin. Microbiol. 8: El Kholy, A., L. W. Wannamaker, and R. M. Krause Simplified extraction procedure for serological grouping of beta-hemolytic streptococci. Apple. Microbiol. 28: Facklam, R. R Streptococci, p In E. H. Lenette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C. 6. Facklam, R. R Isolation and identification of streptococci. Part IV. Agglutination grouping. Department of Health, Education and Welfare. Center for Disease Control, Atlanta, Ga. 7. Facklam, R. R., R. C. Cooksey, and W. C. Wortham Evaluation of commercial latex agglutination reagents for grouping streptococci. J. Clin. Microbiol. 10: Facklam, R. R., J. F. Padula, L. G. Thacker, E. C. Wortham, and B. J. Sconyers Presumptive identification of group A, B, and D streptococci. Appl. Microbiol. 27: Finch, R. G., and I. Phillips Serological grouping of streptococci by a slide coagglutination method. J. Clin. Pathol. 30: Lancefield, R. C Serological differentiation of human and other groups of hemolytic streptococci. J. Exp. Med. 57: Lue, Y. A., 1. P. Howitt, and P. D. Ellner Rapid grouping of beta-hemolytic streptococci by latex agglutination. J. Clin. Microbiol. 8: Matthieu, D. E., Jr., B. L. Wasilauskas, and R. A. Stallings A rapid staphylococcal coagglutination technique to differentiate group A from other streptococcal groups. Am. J. Clin. Pathol. 72: Rantz, I. A., and E. Randall Use of autoclaved extracts of haemolytic streptococci for serological

5 VOL. 12, 1980 grouping. Stanford Med. Bull. 13: Sliflcin, M., C. Engwall, and G. R. Pouchet Direct-plate serological grouping of beta-hemolytic streptococci from primary isolation plates with the Phadebact Streptococcus Test. J. Clin. Microbiol. 7: Slifkin, M., and G. R. Pouchet-Melvin Evaluation of three commercially available test products for serogrouping beta-hemolytic streptococci. J. Clin. Microbiol. 11: Swanson, J., K. C. Hsu, and E. D. Gotschlich MICRONITROUS ACID EXTRACTION AND PHADEBACT 545 Electron microscopic studies on streptococci. I. M antigen. J. Exp. Med. 130: Swift, H. F., A. T. Wilson, and R. C. Lancefield Typing group A hemolytic streptococci by M precipitin reactions in capillary tubes. J. Exp. Med. 78: Watson, B. K., R. C. Moellering, Jr., and L. J. Kunz Identification of streptococci: use of lysozyme and Streptomyces albus filtrate in the preparation of extracts for Lancefield grouping. J. Clin. Microbiol. 1: