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

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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 diagno- sis 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.
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