Streptococcal Erythrogenic Toxin Genes: Detection by Polymerase Chain Reaction and Association with Disease in Strains Isolated in Canada from 1940 to 1991 S

JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1992, p. 3127-3131 Vol. 30, No. 12 0095-1137/92/123127-05$02.00/0 Copyright C 1992, American Society for Microbiology Streptococcal Erythrogenic Toxin Genes: Detection by Polymerase Chain Reaction and Association with Disease in Strains Isolated in Canada from 1940 to 1991 S. D. TYLER,1* W. M. JOHNSON,1 J. C. HUANG,' F. E. ASHTON,' G. WANG,1 D. E. LOW,2 AND K. R. ROZEE' Bureau ofMicrobiology, Laboratory Centre for Disease Control Tunney's Pasture, Ottawa, Ontario KJA OL2,1 and Department ofMicrobiology, Mount Sinai Hospital, Toronto, Ontanio M5G lXS,2 Canada Received 10 July 1992/Accepted 15 September 1992 The presence of genes encoding pyrogenic exotoxins type A (spe4), B (speB), and C (speC) and streptolysin 0 (slo) was determined by the polymerase chain reaction (PCR) to target specific sequences in 152 strains of group A streptococci. These included reference strains, representative M and T type strains, and strains associated with scarlet fever and pharyngitis collected between 1940 to 1991 and included strains from patients with severe invasive streptococcal infections. PCR amplicons were detected by agarose gel electrophoresis, and specificity was established by restriction fragment analysis. The frequency of occurrence for each target gene among all strains tested was 33.6% for speA, 99.3% for speB, 28.9%1o for speC, and 100% for slo. Strains of non-group A streptococci, recognized toxigenic bacterial pathogens, and pneumolysin-producing Streptococcus pneumoniae strains were negative for all targeted gene sequences. Detection limits in the PCR were found to be 100 pg of total nucleic acids for the speB and speC genes and 1 ng for the speA and slo genes. Isolates associated with scarlet fever, pharyngitis, and severe invasive infections showed statistically significant differences in the presence of speA, with scarlet fever strains having the highest association (81.3%), severe infections the next highest association (42.9%o), and pharyngitis the lowest association (18.4%). Although no significant differences were observed in speC frequencies in isolates associated with the three disease categories, a genotype of speB slo was significantly higher in isolates associated with pharyngitis (54.1%) than in strains associated with scarlet fever (18.8%) or severe invasive disease (23.8%). Streptolysin 0 targets were present in all ofthe isolates tested, and only a single strain (T-11-M-11) was devoid of targeted speB sequences, thereby demonstrating that neither speB nor slo is associated with any particular clinical presentation.

Since 1985 there has been an apparent increase in the pyrogenic exotoxin type B (SPEB), encoded by speB, has incidence of invasive streptococcal infections (i.e., bacte- been cloned from the genome of Streptococcuspyogenes (2), remic) (11, 30), severe invasive group A streptococcal infec- but the genetic mechanisms involved in production have not tions (2-5, 8-10, 26, 32-35), and rheumatic fever (20). been well characterized. Several of the patients have presented with a disease remi- Streptolysin 0 (SLO), a thiol-activated toxin, is another niscent of the staphylococcal toxic shock syndrome, and putative virulence factor in streptococcal infections (23). hence, it was termed streptococcal toxic shock-like syn- Although SLO shares some structural homology with pneu- drome (1, 7, 26). In previous years, it was thought that the molysin, listeriolysin, and other hemolysins from diverse introduction of effective antimicrobial therapy and generally genera and species, lack of DNA sequence homology en- improved public health conditions had reduced both the sures the observed specificity of hybridization probes (22). incidence and the severity of these infections (27). An In the present study, we determined the specificities and increase in the occurrence of specific virulence factors, sensitivities of PCR protocols developed to target sequences particularly streptococcal pyrogenic exotoxin type A in the speA, speB, speC, and streptolysin 0 (slo) genes. In (SPEA), has been linked to the apparent resurgence of more addition, the frequency of these genetic elements in strains severe disease (4, 28, 33, 34). collected over five decades was assessed by the polymerase The three erythrogenic toxins produced by group A strep- chain reaction (PCR) and was related to their occurrence in tococci are believed to be associated with pyrogenicity and specific streptococcal disease categories of differing sever- erythematous skin reactions in addition to various immuno- ity. logical and cytotoxic effects. Physicochemically they are very similar in molecular mass, ranging from 24 to 29 kDa for the cloned toxin gene products (2). Because two of the MATERIALS AND METHODS toxins, SPEA and streptococcal pyrogenic exotoxin type C (SPEC), can be transferred by lysogenic conversion (2, 18, Bacterial strains and culture media. A total of 152 strains of 42), mobile genetic elements could account for changes in group A streptococci were investigated. Seven were refer- the frequencies of occurrence of genes encoding both SPEA ence strains (CS-24, 86-858, T-18P, 594, Bell, NY-5, and (speA) and SPEC (speC) in clinical isolates. Streptococcal Wilson), 10 were type strains (T-1-M-1, T-8, 2 strains of T-19, T-11, T-22, T-27, T-47, M-10, M-19, M-47), and 135 were clinical isolates with documentation of clinical diagnosis dating back to 1940. These were collected by the Respi- * Corresponding author. ratory Infections Section, National Laboratory for Bacteri- 3127 3128 TYLER ET AL. J. CLIN. MICROBIOL.

TABLE 1. Base sequences and predicted amplicon product sizes for the pyrogenic exotoxin and streptolysin primers

Toxin Primers (5'-3') withinLocationgene Ampliconsize (bp) forEnzymeconfirmationused Restrictionlengthsfragment(bp) SPEA-1 act taa gaa cCa aga gat gg 351-370 SPEA-2 ctt tat tot tag gta tga ac 684-703 353 Hinfl 142, 211 SPEB-1 gte aac atg cag cta cag ga 714-733 SPEB-2 aat acC aaC ate agC Cat ea 951-970 257 Hinfl 53, 204 SPEC-1 aag tga Ctc taa gaa aga ca 231-250 SPEC-2 ttg agt atC aat gtt taa tg 341-360 130 NsiI 52, 78 SLO-1 aat atC aac aCt aCa CCa gt 661-680 SLO-2 ctg ttg aaa Cat tgg Cat ag 861-880 220 AluI 75, 145

ology, and were stored lyophilized in 10% skimmed milk. possess all of the targeted genes were adjusted to a concen- Twenty-one of the strains included in the present study were tration of 200 ,ug/ml, and serial 10-fold dilutions were made associated with recent cases of severe invasive group A in TE buffer prior to use as the template in the PCR. To streptococcal disease with a 52% fatality rate and have been avoid possible amplicon carryover, the procedures of Kwok described previously (5). The group A streptococcal strains and Higuchi (24) were adopted. were serogrouped (25) and were then T typed (13) and M Restriction endonuclease digestion. Aliquots of 10 ,ul of the typed (36) by using sets of antisera produced in our labora- amplified fragments recovered after PCR were subjected to tory. Streptococcal strains were grown in Todd-Hewitt broth restriction endonuclease digestions by using Hinfl, NsiI, or (Oxoid, Unipath Canada, Nepean, Ontario, Canada) at 37°C AluI (GIBCO-BRL, Burlington, Ontario, Canada) under the overnight in a candle jar prior to nucleic acid (NA) extrac- conditions recommended by the manufacturer. Hinfl was tion. Strains from other genera included toxigenic Esche- predicted to digest the speA amplicon into 142- and 211-bp richia coli, Shigella dysenteriae type 1, Listeria monocyto- fragments and the speB amplicon into 53- and 204-bp frag- genes, Streptococcus pneumoniae, Staphylococcus aureus, ments. NsiI was predicted to digest the speC amplicon into and Vibrio cholerae as described previously (19, 29). 52- and 78-bp fragments. AluI was predicted to yield 75- and NA isolation and PCR. Cells were pelleted from 5 ml of 145-bp fragments from the slo amplicon. The digested sam- 18-h cultures at 1,800 x g for 10 min (IEC Centra 7R ples were analyzed by standard submarine gel electrophore- centrifuge), resuspended in phosphate-buffered saline (pH sis by using 2% agarose gels. 7.4), and lysed by vortexing in the presence of glass beads (31). The NAs were extracted with phenol-chloroform, pre- RESULTS cipitated with ethanol, subsequently dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]), and adjusted to a Specificities and sensitivities of oligonucleotide probes. The concentration of 2 ,ug/ml. Aliquots of 10 ng were amplified by results of primer-directed amplification of the targeted seg- PCR by previously described procedures (29). Table 1 de- ments for speA, speB, speC, and slo genes are represented in scribes the specific oligonucleotide primer pairs used in the Fig. 1. When reference strains of group A S. pyogenes were PCR. The oligonucleotide primer pairs were prepared by the tested for the presence of erythrogenic exotoxin gene tar- Oligonucleotide Synthesis Laboratory, Queen's University, gets, amplicons of the predicted sizes were observed, as Kingston, Ontario, Canada. The primers were designed by summarized in Table 2. Reference strains known to be computerized sequence analysis (6) of the published nucle- phenotypically negative for SPEA and SPEC were always otide sequences for speA (39), speB (15), speC (12), and slo negative for the corresponding gene target in the PCR. The (23) and target sequences within the coding regions of the predicted amplicon with the SPEB primers was seen in all respective genes. It has been shown that SPEB and strepto- but one type strain of T-11-M-11. No differences between coccal protein precursor (SPP) share extensive homologies the digestion patterns of SPEB amplicons from phenotype- and are considered by some to be variants of the same positive and -negative strains were observed. The region protein (15, 37). To ensure the specificity of the protocol for targeted by the SLO primers was also found to be present in SPEB, a potential model of the SPP gene was constructed by all strains tested. No specific amplification was seen with using the minimum number of base substitutions in the any of the primer pairs when non-group A streptococci or SPEB sequence which would result in a coding sequence for other representative pathogenic organisms were tested. The the SPP protein. The primer SPEB-a was then targeted to a levels of sensitivity for these assays were found to be 100 pg region where the SPP gene would contain a minimum of a of total NAs for speB and speC and 1 ng for speA and slo. In 40% mismatch in its corresponding sequence. Under the view of the significant sequence relationship between mature stringent conditions used in this protocol, it seems unlikely SPEA and S. aureus enterotoxin B (seb) (17), S. pyogenes that this primer would bind to the SPP sequence. In addition, NY-S, which is positive for SPEA, SPEB, and SPEC, was the restriction endonuclease Hinfl was chosen such that it tested in the PCR by using primers specific for S. aureus seb would not digest an amplicon originating from SPP. The PCR (19) and were found to be negative. Similarly, NAs from was performed in a 50-pl reaction mixture by using only one strains of enterotoxin B-producing S. aureus were negative primer pair under the following conditions for 30 cycles: in the PCR when all streptococcal exotoxin primers were denaturation for 2 min at 94°C; annealing ofspeA, speB, and used. slo primers for 2 min at 55°C and speC primers for 2 min at As a strategy to confirm and validate amplicon integrity, 45°C; and primer extension for 1 min at 72°C with an specific restriction endonuclease sites were identified in the automatic increase of 5 s per cycle. To test the sensitivity of targeted amplicons and restriction fragment length polymor- the PCR procedure in detecting the target gene fragments, phism patterns were analyzed. Digestions were performed NAs from a reference strain of S. pyogenes (NY-5) known to with Hinfl, NsiI, or AluI on aliquots of the amplicons VOL. 30, 1992 PCR DETECTION OF STREPTOCOCCAL EXOTOXIN GENES 3129

TABLE 3. Frequency of erythrogenic exotoxin genes in group A streptococcal strains associated clinically with pharyngitis, scarlet fever, and severe invasive disease % of isolates with the Clinical syndrome Era of following genotypea: (no. of isolates) isolation speC speA and speB speC only Pharyngitis (98) 1983-1990 18.4 30.6 3.lb 54.1c4 Scarlet fever (16) 1940-1941 81.3b,c 25.0 25.0b 18.8c,d Severe invasive 1987-1991 42.9b.c 33.0 0 23.8C disease (21) a All clinical isolates possessed speB and slo; all significant differences between the frequency of genotypes in each clinical syndrome are noted. b p < 0.01. c p < 0.05. d p < 0.05. FIG. 1. Agarose gel electrophoresis showing typical PCR amplification fragments and restriction fragment length polymorphisms for targeted regions of the speA, speB, speC, and slo genes of group observed in the clinical strains were as follows: speB alone, A S. pyogenes. Lanes a and n, 123-bp ladder (Bethesda Research 45.2%; speA and speB, 24.4%; speB and speC, 25.2%; and Laboratories); lanes b and c, S. pyogenes NY-S with SPEA primers; speA, speB, and speC, 5.2%. The most predominant sero- lanes d and e, S. pyogenes NY-S with SPEB primers; lanes f and g, type encountered was T-1-M-1 (38.2%), and of these, 82.8% S. pyogenes NY-S with SPEC primers; lanes h and i, S. pyogenes of T-1-M-1 strains were of the speB slo genotype and only NY-S with SLO primers; lanes c and e, amplicons from lanes b and 17.2% were of the speA speB slo genotype. d digested with Hinfl; lane g, amplicon from lane f digested with Table 3 summarizes the data showing the frequency of NsiI; lane i, amplicon from lane h digested with AluI; lanes j to m, erythrogenic exotoxin genes in three categories of group A S. pyogenes DC11435 (T-11-M-11) with SPEA, SPEB, SPEC, and SLO primers, respectively. streptococcal infections. Statistically significant differences between gene frequencies were determined by computing the interaction chi-square. generated in the PCR by using NAs from S. pyogenes NY-5 and primers for speA, speB, speC, and slo (Fig. 1). The DISCUSSION restriction fragment length polymorphism patterns obtained In the present study, we examined strains of S. pyogenes experimentally were identical to those predicted from the that are clinically associated with three categories of infec- published nucleotide sequences of the targeted areas of the tions: pharyngitis, scarlet fever, and severe invasive disease. speA, speB, speC, and slo genes. The PCR results for strain Pharyngitis and scarlet fever groups were represented by DC11435 (T-11-M-11) appear in lanes j through m of Fig. 1, isolates chosen from our culture collection, which encom- in which the exotoxin primers for speA, speB, speC, and slo, passes a 51-year time span, and their association with the respectively, were used. The results indicate that the exo- clinical diagnosis forms part of our documentation of these toxin target present is speA (Fig. 1, lane j) in this NA. This strains. Most previous toxin studies, which have been lim- is the only strain in the present study that was negative for ited to single groups of clinical patients (16), are based on the speB gene target (Fig. 1, lane k), and these observations traditional phenotypic analyses (14) or hybridization tech- were confirmed by multiple extractions of several colonies of niques (16, 40, 41). In their study of 34 strains of group A the same strain, with identical results. streptococci from clinically well-documented toxic shock- Distribution of genotypes. The results indicated that the like syndrome, Hauser et al. (16) observed that speA is frequency of detection of each toxin gene among all strains associated with toxic shock-like syndrome in 85% of strains, tested was 33.6% for speA, 99.3% for speB, 28.9% for speC, even though only 53% of the strains elaborated the gene and 100% for slo. The distributions of exotoxin genotypes product. They suggested that SPEA production may be down-regulated under in vitro growth conditions in M type 1 organisms and could not rule out the possibility that the TABLE 2. Distributions of speA, speB, and speC targeted gene toxin was not expressed in vivo. Because the in vitro fragments in reference strains of group A streptococci stability of the speA genotype was examined and found to be stable even after 20 passages (16), we consider the most Erythrogenic exotoxin meaningful comparisons between currently published stud- designation Phenotype" genotype" by PCR ies relate to gene frequencies and specific genotypes deter- speA speB speC mined either by hybridization or by gene amplification

CS-24 Negative - + techniques. Further studies will be necessary to elucidate 86-858 SPEB - + - completely the expression of pyrogenic exotoxin genes in T-18P SPEC - + + group A streptococci. 594 SPEA + + _ The PCR protocols developed in the present study for the Bell SPEA SPEB + + detection of genes encoding pyrogenic exotoxins and SLO NY-5 SPEA SPEB SPEC + + + are both sensitive and specific for their targeted gene seg- Wilson SPEA SPEC + + + ments. Specificity was demonstrated by the equivalence of aStrains and phenotypes were kindly provided by P. M. Schlievert. the predicted and observed restriction fragment length poly- "All reference and type strains possessed slo. morphism patterns of digested amplicons (Table 1, Fig. 1), the 3130 TYLER ET AL. J. CLIN. MICROBIOL. observed genotypes of reference streptococcal strains (Table the report by Yu and Ferretti (41) and 81.3, 100, and 25%, 2), and the consistently negative results obtained in PCR tests respectively, in the present analysis. The only major differ- with NAs from a variety of toxigenic pathogens, including ences observed between the two studies relate to the lower organisms known to produce thiol-activated hemolysins (23) frequencies in the present study for speC in both general or enterotoxins with known sequence relationships to the pharyngitis group A and scarlet fever isolates and a consid- erythrogenic toxin genes (17). Although the observed detec- erably higher incidence of speA in scarlet fever strains. tion limits were not identical for all the targeted sequences, These differences are unrelated to the numbers of subcul- the range was similar to that observed in other PCR protocols tures in various laboratories since Kaplan et al. (21) have which target bacterial toxin genes (19, 29). shown that expression of genes for these toxins remains In the present study, all clinical group A streptococci stable after several passages. Phenotypic analysis of 40 investigated were found to possess targeted gene segments scarlet fever strains in a previous study (14) failed to for both speB and slo, and a single type strain was negative demonstrate SPEA production by any of the strains, SPEB for the speB target. Representative strains of group B production by 70% of strains, and SPEC production by 40% streptococci and S. pneumoniae were negative for both speB of strains. Our results indicate that speA appears in conjunc- and slo, thereby further validating the specificities of these tion with speC significantly more frequently in the scarlet probes for group A virulence factors. It has been established fever strains (25%) than in the pharyngitis strains (3.1%) (P that SPEB is a variant of the streptococcal proteinase < 0.01). In addition, the absence of the speA and speC genes precursor (15, 37), and with the exception of one strain, our was significantly more frequent in the general pharyngitis results agree with other studies reporting speB in all isolates strains (54.1%) than in the scarlet fever or toxic shock-like of group A streptococci (16, 41). Since speB and slo were syndrome strains (18.8%). The same trend in gene distribu- present in strains isolated from patients in all clinical cate- tion between these two genotypes was reported by Yu and gories, their presence or absence may not be associated with Ferretti (41). In general, the trends in gene distribution increased virulence. Similarly, speC is present in the same reported by Yu and Ferretti (41) with hybridization probes proportion in strains isolated from patients in all clinical were similar to those encountered in the present study. categories and, except in association with speA and scarlet Although it was not our intent to study the occurrence of fever, by itself may not confer enhanced virulence. the speA and speC genes among group A streptococcal M- Several reports have discussed the recent reemergence of and T-serotype strains, we observed that 57 of 98 strains in the SPEA toxin and the speA gene in group A S. pyogenes the general group A category were of M type 1 and T type 1, and its possible role in the increase in virulence of some of none of which contained the speC gene and only nine of which these isolates (4, 28, 33, 34). In the present study of 135 contained the speA gene. A recent report from the Centers for clinical strains, as summarized in Table 3, it was found that Disease Control, Atlanta, Ga. (32), demonstrated that the there was a significant (P < 0.01) decrease in the prevalence proportions of M types 1, 3, and 18 increased significantly in of speA in strains associated with pharyngitis in the 1980s a study of 5,193 strains sent to the Centers for Disease Control (18.4%) compared with the prevalence of speA in strains between 1972 and 1988. These M types were more likely to be associated with scarlet fever in the early 1940s (81.3%). In invasive, to cause fatal infection, and to occur in a cluster of strains associated with severe invasive disease, the fre- infections than were other types. We did not have a large quency of occurrence of this gene increased to 42.9%, with enough sample of strains to draw any major conclusions significant differences between strains isolated from patients concerning the frequencies of spe genes in specific serotypes. in all three disease categories. A high frequency of speA In the severe invasive category, 8 of the 21 strains were carriage and SPEA production by isolates associated with M-1-T-1. Results of multilocus enzyme electrophoresis are severe invasive disease has been documented by several suggestive of two major clones associated with the majority of laboratories by both phenotypic (16, 33) and genotypic (16, severe invasive disease isolates. One clone is essentially 41) methods. Specific comparisons of gene frequencies are limited to M type 1 and the second is limited to M type 3 (28). possible only with the isolates from patients with severe Data suggest that changes in the epidemiology of group A invasive disease in our study and that of Hauser et al. (16), streptococcal disease may be related to changes in the distri- since the genotypic hybridization analysis performed by Yu butions of M types that cause infection (32). and Ferretti (41) did not include isolates from patients with It appears from the results of this and several other studies these types of infections. Gene frequencies of 42.9, 100, and that speA is usually associated with group A streptococcal 33.3% for speA, speB, and speC, respectively, determined strains isolated from patients with severe invasive disease. by PCR for invasive isolates in our study of 21 strains Because this pyrogenic exotoxin may not always be present compares with gene frequencies of 85, 100, and 21%, respec- in isolates from patients with severe invasive disease, other tively, in the 34 similar strains determined by hybridization risk factors including those of the host are also likely to be in the study by Hauser et al. (16). important (34, 38). The gene frequencies determined in the present study can be compared with those documented by Yu and Ferretti (41) ACKNOWLEDGMENTS for the similar clinical subgroups of pharyngitis group A and scarlet fever isolates. For pharyngitis group A isolates, We gratefully acknowledge P. M. Schlievert for providing reference strains and the Genetics Computer Group, University of Wiscon- speA, speB, and speC were present at 14.9, 100, and 50.8%, sin, for extensive use of the Sequence Analysis Software Package. respectively, in the hybridization study (41) and 18.4, 100, G.W. is a Natural Sciences and Engineering Research Council of and 30.6%, respectively, in the current PCR-based investi- Canada Visiting Fellow. gation. A genotype of speA speC occurred in fewer than 5% of strains in both studies, and neither speA nor speC (speB REFERENCES only) was encountered in 39.4% of isolates in the study of Yu 1. Bartter, T., A. Dascal, K. CarroUl, and F. Curley. 1988. 'Toxic and Ferretti (41) and in 54.1% of isolates in the present strep syndrome' a manifestation of group A streptococcal study. For scarlet fever isolates, gene frequencies were 52.6, infection. Arch. Intern. Med. 148:1421-1424. 100, and 48.7% for speA, speB, and speC, respectively, in 2. Bohach, G. A., D. J. 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