Received 15 November 1996/Returned for modification 3 February 1997/Accepted 17 February 1997

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1 JOURNAL OF CLINICAL MICROBIOLOGY, May 1997, p Vol. 35, No /97/$ Copyright 1997, American Society for Microbiology Survey of emm Gene Sequences and T-Antigen Types from Systemic Streptococcus pyogenes Infection Isolates Collected in San Francisco, California; Atlanta, Georgia; and Connecticut in 1994 and 1995 BERNARD BEALL,* RICHARD FACKLAM, THERESA HOENES, AND BEN SCHWARTZ Childhood and Respiratory Diseases Branch, Centers for Diseases Control and Prevention, Atlanta, Georgia Received 15 November 1996/Returned for modification 3 February 1997/Accepted 17 February 1997 The variable 5 emm (M-protein gene) sequences and T-antigen types were determined from 340 systemic group A streptococcal (GAS) isolates taken from hospitalized patients in San Francisco, Calif.; Atlanta, Ga.; and Connecticut in 1994 and Eighty percent of these isolates had emm sequences and T-antigen types in agreement with previously recorded M- and T-antigen associations. Most of the remaining strains either were T nontypeable (11%) or contained emm genes encoding M proteins for which T-antigen associations have not been made (6%). One newly encountered emm gene, designated ST2974, from each of 13 isolates had the T type 8/25/Imp19. Another new emm gene, ST2967, from 8 of 11 isolates was T nontypeable. Six other unique emm gene sequences from seven isolates were encountered. Sequencing of the variable region of the emm gene of GAS isolates (emm typing) is effective for surveying the sequence variability of the M virulence protein, and combined with T typing, emm typing is useful for monitoring GAS strain diversity. The emm gene of group A streptococcal (GAS) isolates encodes a major virulence factor of these important pathogens, the M protein (1, 7, 11, 13, 14). The variable sequences of surface-exposed amino termini of M proteins appear to provide the basis for identifying about 80 different serological M types (1, 7, 11, 14, 15), each of which is often associated with specific T-antigen patterns (5, 6, 10, 17). Combined M- and T-protein antigen typing has proved to be useful over the years for epidemiologic study of GAS isolates; however, M typing reagents are not widely available and are difficult to prepare. In addition, the process of obtaining unambiguous M typing results can be notoriously difficult. This is further complicated by the difficulty of obtaining high-titer anti-m sera against opacity factor (OF)-positive strains (5). In many laboratories the cost of replenishing diminishing M typing serum stocks is prohibitive. Perhaps more importantly, the remarkable numbers of established M specificities and M-nontypeable strains seriously limits the usefulness of serologic M typing. It is impractical to continue to maintain a comprehensive stock of M typing sera. Recently, we demonstrated the usefulness of emm gene sequence analysis combined with T-antigen typing and OF phenotyping for the routine typing of group A streptococci (2). Using Lancefield reference strains for most of the known M types, we showed that for the majority of these reference strains there was an excellent correlation between previously determined emm sequence and M types. With a limited set of clinical GAS isolates from normally sterile body sites, we also found a good percentage of agreement between emm sequence types and common M and T typing associations. For the work presented here we applied this methodology for typing 340 systemic GAS isolates taken from hospitalized patients in three population centers in 1994 and * Corresponding author. Mailing address: Centers for Disease Control and Prevention, Mailstop C02, 1600 Clifton Rd., NE, Atlanta, GA Phone: (404) Fax: (404) address: beb0@ciddbd2.em.cdc.gov. MATERIALS AND METHODS Strains. All 340 isolates used for this study were isolates from populationbased collections of blood from different hospitals throughout the three survey areas. The survey areas included metropolitan Atlanta, Ga. (32 hospitals); metropolitan San Francisco, Calif. (40 hospitals); and Connecticut state (35 hospitals). All strains except for the serotypeable M1 and M3 isolates from the Atlanta area were subjected to emm sequence analysis; the serotypeable M1 and M3 isolates from Atlanta were not included among the 340 strains. Serologic typing and OF determination. Serologic M types, T types, and OF reactions were determined as described previously (16, 17). M typing antisera were available for types 1, 2, 3, 4, 5, 6, 8, 11, 12, 14, 15, 17, 18, 19, 23, 24, 25, 26, 27, 29, 30, 31, 32, 33, 36, 37, 38, 39, 40, 41, 42, 43, 46, 47, 49, 51, 52, 53, 54, 55, 56, and 57. PCR. Lysates of the GAS isolates were prepared with mutanolysin and hyaluronidase as described previously (2). Primers 1 and 2 were used in PCRs as described previously (24). Sequence analysis. Approximately 60 ng each of up to 36 PCR products was sequenced with primer emmseq2 (5 -TATTCGCTTAGAAAATTAAAAACA GG) with the dye terminator mix (Applied Biosystems, Foster City, Calif.) and was subjected to automated sequence analysis on a 373 or 377 DNA sequencer (Applied Biosystems) as described previously (2), except that an annealing temperature of 55 C was used. DNA sequences were subjected to homology searches against the bacterial DNA database with Genetics Computer Group software (Wisconsin package, version 8). For these comparisons, single in-frame deletions or insertions of up to seven codons were not taken into account. Such in-frame deletions and insertions occurred at a frequency of less than 3% among the sequences represented. Sequences were given the GenBank emm designations indicated in Table 2 if 95% identity was observed over at least the first 160 bases of the sequence, although in the majority of cases, 300 to 500 bases of accurate sequence was obtained. Shorter ( 220-base) sequences were used in many cases due to two factors. In some instances, long GenBank sequences were not available. Also, in a minority of cases, longer sequences were not as accurate. The GenBank accession numbers for the emm and emm-like genes have been presented previously (2, 4, 12, 19, 20, 24 26). Those for the sequences emmst2967 and emmst2974 which were obtained in this study are given below. Seven sequences from this study that have not been submitted to GenBank are available upon request and include those of emmst2980 and six other unique emm genes. Definitions. M type strains that have had type-specific precipitating antibody with bactericidal activity made by more than one reference laboratory are represented by M types M1 to M81. The corresponding emm gene sequences are designated in Table 2 as emm1 to emm80. Provisional M types were obtained from strains that have been observed to have type-specific precipitating antibody with bactericidal activity made by one laboratory, but these types remain provisional because they have not been confirmed by two reference laboratories. The corresponding gene sequences (26) encountered in this study are designated in Table 2 as emmpt180, emmpt2110, emmpt4245, and emmpt

2 1232 BEALL ET AL. J. CLIN. MICROBIOL. TABLE 1. T types historically associated with M or emm types encountered in these studies a M type OF T pattern(s) b , 13, 3/13, 3/13/B , 4/28 5 5, 5/27/ , 9/ , 11/ , 11/ , 3/13/B3264, 4/ , , 4/ /13/B3264, 3/ /13, 3/13/B / , 14/ /13/B /3/ , 8/25/Imp , 11/12, , 9/11/ /13/B , 8/25/Imp , 11/ , 13/B pt180 5/27/44 pt2110 3/13/B3264 pt pt2841 4, 4/28, 28 st64/14 c 6 st2974 c 8/25/Imp19 a T types are from extensive surveys of thousands of strains typed in previous studies (7 9) and approximately 20,000 strains typed in this laboratory from 1966 to b Any given M type may sometimes include T-nontypeable strains. c T type specificity provided by the isolates examined in this work and several isolates from other locations (2a). Sequence analysis of the type genes was performed with the emm-specific primers described above, and this sequence was found to be less than 90% identical to the gene sequences of the M type strain (emm genes) and provisional M type strains (emmpt genes). These gene sequences are designated in Table 2 as emmst genes. Nucleotide sequence accession numbers. The sequences of emmst2967 and emmst2974 obtained in this study have been given GenBank accession numbers U50337 and U50338, respectively. RESULTS T and M typing correlates. Table 1 presents the M and T typing associations obtained by typing more than 20,000 strains at the Centers for Disease Control and Prevention (CDC) during the years 1966 to Although these are common M and T type associations among GAS strains, not all strains of the same M type will have these same T type associations (5, 10). All Atlanta, San Francisco, and Connecticut GAS isolates were emm typeable. All (340 of 340) of the isolates in this study yielded an emm-specific PCR fragment suitable for sequence analysis, the results of which are presented in Table 2. Prevalence of previously sequenced emm genes. Overall, 308 (90.6%) of 340 isolates in this study had 5 emm sequences 95% identical to 1 of 37 emm or emm-like genes in GenBank. For most of these sequences, this high level of identity actually TABLE 2. emm sequences of GAS isolates from patients in San Francisco, Atlanta, and Connecticut with systemic infections a No. of isolates emm No. of isolates SF AT b CT M typeable Conform No. of isolates for which emm (M) or T typing correlates c : Do not conform Are TNT st st NA NA pt st64/ pt pt stns NA NA 1 2 enn NA NA 0 2 st NA NA pt Unique NA NA 1 a Abbreviations: NT, nontypeable; SF, San Francisco, AT, Atlanta; CT, Connecticut; NA, not available. b M typeable M1 (n 44) and M3 (n 30) isolates from Atlanta were not included. c Conform, the emm sequence agrees with previously observed M- and T- antigen combinations; do not conform, a new T-antigen type was observed for this M (emm) type; TNT, T nontypeable. extended to 200 to 450 bases, without diminishing. The sequences of 280 (82%) isolates were 95% identical to the sequence of 1 of 31 standard M type reference strain emm genes, and the sequences of 17 isolates (5%) were 95% identical to the sequence of 1 of 4 provisional M type strain emm genes (designated with just numbers and pt followed by numbers, respectively, in Table 2). The remaining 11 of these 308 isolates had emm sequences that were highly similar or identical to those of st64/14 (7 isolates) (4), enn63 (2 isolates) (25), or stns5 (2 isolates) (20). The st sequences are not linked with known M typing specificities, and the enn genes are de-

3 VOL. 35, 1997 emm SEQUENCE SURVEY OF SYSTEMIC S. PYOGENES ISOLATES 1233 scribed as being distinct from emm genes on the basis of their relative positions within the vir locus and sequence differences (25). Prevalent emm genes. In agreement with the M typing surveillance done by CDC over the past 15 years (21), the prevalent emm sequences encountered in these systemic isolates were emm1 and emm3, which accounted for about 24% of the surveillance isolates. As with the San Francisco and Connecticut surveys, M1 and M3 isolates were also common in the Atlanta area, accounting for 44 and 30 isolates, respectively, of a total of 166 isolates (data not shown). However, these 74 M1 and M3 strains were not included in this emm sequence survey. For this work, only Atlanta T1 and T3 M-nontypeable strains were subjected to emm sequence analysis. All three M-nontypeable T1 strains and the one M-nontypeable T3 strain (Table 2) clearly had emm1 and emm3 sequences nearly identical to those of their respective GenBank entries. On the basis of the relationship established between published emm sequences and M serology (2, 12, 19, 24, 26), specific M typing sera were not available for 50% of the isolates in this study (see sera available for use in Materials and Methods). Other than emm1 and emm3, there was no marked preponderance of a given emm sequence, with 11 emm sequences each being present in 10 or more isolates (Table 2). Besides emm1 and emm3, the most commonly encountered sequences in isolates from San Francisco and Atlanta were emm76 (11 isolates) and emm59 (12 isolates), respectively. In isolates from Connecticut, the two most frequently encountered sequences were emm28 and emm12, each of which was present in 10 isolates. It was interesting to find that two common emm sequences had not been previously documented. One of these emm sequences, st2967, was found in 11 isolates (10 from San Francisco and 1 from Atlanta), 8 of which were T nontypeable (Table 2). Comparisons of this sequence with the corresponding regions of other emm genes showed a high level of sequence identity within the deduced signal sequence-encoding region, which is highly conserved in emm genes (part of the signal sequence is shown in Fig. 1), followed by a more variable region putatively encoding the N terminus of the mature protein (Fig. 1). A homology search showed the amino acid sequence of the deduced ST2967 variable region to be quite similar to the emm2 gene product (Fig. 1A), with the two genes sharing almost 85% DNA sequence identity (data not shown). Another common emm sequence that had not been previously described was st2974, which was found in seven isolates from San Francisco, five isolates from Atlanta, and one isolate from Connecticut (Table 2). Since all 13 of these isolates shared the common T pattern, T8/25/Imp19, there is a strong possibility that these strains are from a common origin. The closest match to the translated amino acid sequence of st2974 was the M27 protein, with about 59% identity over 91 residues (Fig. 1). Overall, more than 11% of the San Francisco isolates contained either st2967 or st2974 emm sequences. Undocumented emm sequences. Besides st2967 (found in San Francisco and Atlanta) and st2974 (found in all three areas), we found a total of seven other undocumented emm sequences. One of these, designated st2980, was found in two isolates from San Francisco. The six other undocumented sequences occurred singly in isolates from San Francisco (two isolates), Atlanta (one isolate), and Connecticut (three isolates) (Table 2). These new emm sequences were less than 85% identical to known emm sequences over the first 210 to 270 bases, even when in-frame deletions and insertions were not taken into account (data not shown). FIG. 1. Amino acid (aa) sequence alignment of the deduced N-terminal sequence of Mst2967 with the M2 protein (A) and Mst2974 with the M27 protein (B). Amino acid 1 in all instances is predicted to occur at approximately residue 16 to 25 of the respective precursor protein leader sequence on the basis of homologies with other M and M-like proteins. The first residue of the mature proteins ( ) is predicted in the same manner. New T and emm gene associations. Only 10 T-typeable, emm-typeable strains listed in Table 2, for which previously recorded M and T associations exist (Table 1), did not conform to these T and M type associations. These new M- and T- antigen combinations are presented in Table 3. Agreement between emm types and M types. One hundred eight (64.3%) isolates (including the 47 M1 isolates and the 30 M3 isolates in Table 2) were M typeable and accounted for 11 different serotypes. The emm sequences from the 108 M-typeable isolates from the three geographic regions correlated very closely to the emm sequences previously determined for the respective M type reference strains. The data are consistent with the previous observation that the 5 emm gene sequence can usually be used to predict the M types of M-typeable strains (2). The data are also consistent with our recent observation (see Discussion) that many of our M typing antisera stocks are of poor quality, since 52 isolates with deduced typeable M proteins were in fact M nontypeable. T-nontypeable strains. A total of 38 isolates (11.2%) were T nontypeable. Twelve of these isolates had emm sequences TABLE 3. New emm gene and T-antigen associations found in this study emm No. of isolates a : SF AT CT T-antigen pattern /25/Imp /13/14/27/ /13/B pt and 8 for SF and CT, respectively. a SF, San Francisco; AT, Atlanta; CT, Connecticut.

4 1234 BEALL ET AL. J. CLIN. MICROBIOL. (those for stns5, st2967, st2980, and unique genes) for which T associations have not previously been made. DISCUSSION The purpose of this study was to survey the genetic diversity of systemic GAS isolates in three population-based collections of isolates by using emm gene sequence analysis and T typing. We did not type M type 1 and M type 3 isolates from Atlanta. We have found little sequence variation among emm1 and emm3 alleles from extensive sequencing of M1 and M3 strains nationwide (data not shown). Others have also noted very little sequence variation among emm1 alleles (26), although significant variations in emm genes conferring the M1 serotype have been found (18). Serologic tests for T- and M-protein antigens have long been the primary methods for the identification and epidemiologic study of GAS isolates. We have observed that the supplementation of this methodology with rapid emm gene sequencing greatly improves the efficiency and accuracy of epidemiologic studies of these important pathogens. Even when one assumes that typing sera for all of the known M types and provisional types are available, many strains that are encountered have new emm genes that encode M proteins with new serologic specificities. In our survey of 340 isolates, we encountered 43 such strains with emm genes encoding M proteins for which serologic specificities have not been determined (st64/14, stns5, st2974, st2967, enn63, st2980, and six other unique emm genes listed in Table 2). It is naive to assume that any stock of M typing sera will suffice in the subtyping of GAS isolates, since new M types undoubtedly will continually surface. Indeed, we have found that the percentage of isolates from normally sterile sites from patients in Brazil, New Guinea, Gambia, Ethiopia, and Malaysia with new emm gene sequences is much higher than the percentage of such isolates found in this study and certain European countries (data not shown). These recent findings are consistent with a previous statement maintaining that there is a higher percentage of M-nontypeable strains from Africa than from Britain (5). The difficulties that we encounter in our laboratory may reflect the difficulties seen by others in keeping comprehensive and high-titer M typing sera. Recently, we tested our typing sera by attempting to M type our M typing reference strains. We found that Lancefield extracts of many strains no longer consistently gave positive results with their respective M typespecific serum, and positive reactions were often very weak. Thus, it appears that many of our stocks of M typing sera may have lost their activity. Unfortunately, time and labor constraints prevent the replenishment of these stocks, and many of the typing sera will no longer be used. Except in certain situations in which confirmatory results are desired, the current system of accurate and rapid 5 emm sequence analysis (emm typing) has largely replaced M serotyping at CDC. While other useful DNA-based techniques for subtyping and genetic analysis of GAS isolates have recently been described (for example, see references 3, 8, 12, 22, and 23), we feel that epidemiologic typing is most meaningful when it is based primarily upon a system most sensitively reflecting M specificity. Although it was not used to generate the data presented here, we have subsequently made emm typing more rapid by highresolution restriction enzyme analysis of sets of emm-specific PCR products from temporally and geographically related isolates sharing the same T and OF patterns. This allows us to presume that many isolates within such sets that have identical emm restriction cleavage profiles have identical emm gene sequences while subjecting only a limited number of each set to emm gene sequence analysis for verification (data not shown). The M protein has been thoroughly established as a primary virulence factor, and conventional M typing throughout the past several decades has provided an excellent basis for emm typing. As we have maintained previously, emm sequencing must be supplemented with other selected approaches, such as T typing, to presumptively identify related GAS isolates. There is extensive proof of genetic divergence among strains sharing the same emm gene sequence (2, 3, 18, 21, 26). For example, in the Atlanta survey, we found different T types among emm11 and emm77 strains (Table 2). In the San Francisco survey, we found different T types among emm75, emmpt4245, and emmst2967 strains (Table 2). In the Connecticut survey, we found different T types among emmpt4245 strains (Table 2). The combination of emm sequence analysis and classical T typing is effective because it complements the large amount of epidemiologic data that have accumulated from serotyping of GAS isolates over the past three to four decades. However, while T typing appears to be rapid and reliable for the majority of GAS strains, our data and those from other studies documenting T-nontypeable strains indicate that other methods of typing will be required to accompany emm sequencing for T-nontypeable strains (Table 2) (2, 5). Methods such as random amplified polymorphic DNA analysis (8) and pulsed-field gel electrophoresis of chromosomal digests (22) are viable alternatives to T typing; however, these methods are more time-consuming and the results are not as easily interpretable. It is possible that the characterization of the gene(s) encoding the T antigen(s) may eventually provide the basis for a simple sequence-based alternative to T typing. Although not shown in this study, OF reaction determination is also useful in typing and was always carried out for the isolates examined in this work. GAS isolates of all standard or provisional M types have primarily been associated with either an OF or an OF phenotype. For the most part we found very good agreement between emm sequences indicating standard or provisional M types and the OF phenotypes previously established for these M(emm) types (data not shown). We found nine previously undocumented emm gene sequences (those of six unique genes and genes st2980, st2967, and st2974). Of particular interest was the prevalence of strains containing emmst2967 and emmst2974 that we found (a total of 11 and 13 isolates, respectively; Table 2). These strains were isolated in different years (data not shown) and in geographically separate locations, indicating the likelihood that these GAS strains are widely distributed in the United States. Given the deduced amino acid sequence homology between the emmst2967 and emm2 products (Fig. 1), it is interesting that, as with one of our emmst2967 strains, serotype M2 GAS isolates are OF positive and the T type is sometimes T8/25/Imp19 (10). For the majority of isolates, the emm gene sequence has proven to correlate well to established M serotypes (2, 12, 26). This emm typing methodology has allowed us to tentatively associate other established M serotypes with previously undocumented T type associations in M-nontypeable strains. This may lead to the discovery of other prevalent strains of established M or emm types. For example, it is possible that the two examples of emm9, T14 systemic isolates found in Atlanta and Connecticut (Table 3) reflect a wide geographic distribution of a specific strain. Recently, serotypes of 16,909 GAS isolates obtained from 1980 to 1990 were reported (5). Of these, 1,244 were M nontypeable, even though these researchers also used the T type and anti-of reaction to deduce the M types (9, 16). In that survey they did not find M type 43 or 56 strains, while in our

5 VOL. 35, 1997 emm SEQUENCE SURVEY OF SYSTEMIC S. PYOGENES ISOLATES 1235 much smaller survey, we found a total of 11 emm43 isolates and 1 emm56 isolate. It is possible that the lack of representation of M43 and M56 isolates in the previous study was due to technical difficulties frequently encountered in M serotyping. Because of the continuing worldwide problem of disease mediated by GAS isolates, the spread and genetic variability of these important pathogens must be monitored. Monitoring of GAS isolate and emm genetic variability should eventually lead to a better understanding of the epidemiology and origins of specific GAS strains. ACKNOWLEDGMENTS We thank Anne Whitney and Jan Pruckner for the use of the automated sequencers in their respective laboratories. We are also grateful to Ruth Franklin, John Elliot, Leslye Brudzinski, and Terry Thompson for serotyping. REFERENCES 1. Beachey, E. H., J. M. Seyer, J. B. Dale, W. A. Simpson, and A. H. 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