New emm (M Protein Gene) Sequences of Group A Streptococci Isolated from Malaysian Patients.

To the Editor: We analyzed the M-type-specific emm gene sequences of 24 random Streptococcus pyogenes isolates from sterile- and nonsterile-site clinical specimens of Malaysian patients. In contrast to isolates in the United States, which rarely have new emm sequences, 6 of these 24 Malaysian isolates had new emm gene sequences, which suggests a large reservoir of group A streptococci expressing new M-type specificities in Malaysia.

The M protein is a surface-exposed principal virulence factor of group A streptococci (GAS) and a potential vaccine candidate. The hypervariable M-type-specific N-terminal portion of the M molecule extends from the cell wall and evokes protective antibodies. Approximately 75 M antigenic types of GAS are recognized, and several provisional types have been proposed (1). Formulation of a universally effective vaccine is complicated by the M-typespecific nature of protective anti-GAS antibodies, temporal and geographic variations in GAS M-type prevalence (2), and lack of information on GAS M types from areas where rheumatic fever and rheumatic heart disease, sequelae of GAS pharyngitis, are endemic (3). The lack of specific M-typing antisera is also a limiting factor in determining type distribution. Recently, Beall and colleagues (4.5) demonstrated that sequence analysis of the hypervariable portion of the emm gene encoding M-type specificity (emm typing) was an alternative when M-typing antisera were not available.

Attempts to type selected Malaysian strains of GAS by M protein status have yielded poor results. Fewer than 16% of strains were typable with standard M-typing antisera (6). The existence of new M types in Southeast Asia was suggested as an explanation. To investigate this possibility, we subjected 27 selected strains (6 from blood, 15 from pharyngitis, 3 from pus, and 3 pharyngeal carrier cultures) collected between January 1994 and December 1996 to emm typing. Initial isolation, serogrouping, T typing, and determination of opacity factor production were performed in Kuala Lumpur, by standard techniques, commercial media, reagents, and antisera (7). Strains were transported to the Centers for Disease Control and Prevention in Atlanta, Georgia, USA, for emm typing, where serogrouping, T typing, and opacity factor determinations were repeated, and emm typing was performed (4,5). DNA sequences were subjected to homology searches against all known emm sequences by Genetics Computer Group Software, Version 9. (Most sequences in this database were found in strains isolated from patients living in Europe and North America.)

Of the 27 cultures analyzed, 24 were GAS, 2 were group G streptococci, and 1 was nongroupable Streptococcus. Ten of the 24 GAS strains were standard emm types emm3, emm12, emm22, emm60, and emm76 (encoding the classic M types M3, M12, M22, M60, and M76, respectively); 4 were the provisional emm types pt180, pt2841, and pt5757; and 3 were previously identified emm sequence types st64/14 and st2034 (GenBank accession numbers X72932 and U74320, respectively). The st2034 sequence, originally identified in children from Papua New Guinea, has also been found in Brazil, California, and Hawaii (B. Beall, R. Facklam, unpub, data). One GAS had a sequence previously found in group G streptococci (emmLG6, accession number U25741). Finally, 6 were of five new emm sequence types discovered in this study (st4529, st4547, st4532, st4545, and st3018, with accession numbers AF060368, AF052426, AF077666, AF077668, and AF077669, respectively). The newly found group A st4545 sequence was more similar to various group G streptococcal emm sequences than to known group A emm sequences. One group G isolate had a previously found group G 5' emm sequence (stLG6, accession number U25741). The nongroupable Streptococcus had an emm sequence previously associated with group L Streptococcus (Beall and Facklam, unpub, data). These results demonstrate the usefulness of emm typing in areas where specific M-typing antisera are not available.

Identifying 6 (25%) of 24 GAS with new emm types provides further evidence of new M serotypes of GAS in Malaysia. The deduced amino acid sequences of the mature hypervariable N termini of ST4529, ST4532, ST4547, and ST3018 ranged from 43% to 82% identity to M proteins of known sequence (data not shown). The M nontypability of these isolates suggests unique serologic specificity. ST4547, ST4532, and ST3018 had 70% to 82% identity over the first 55-variable-region amino acids, with their closest matching known M proteins (M70, M27, and M22, respectively), but whether antibodies against any of these proteins would cross-protect against strains expressing these M proteins is unknown. Even though the M70 protein is 70% identical over its first 50 variable N terminal amino acids to the M33 protein, antibodies against the M70 and M33 proteins do not cross-protect, which suggests that no cross-protection would occur. The new deduced M protein with the lowest similarity to any known M protein was ST4529, whose closest match had a 43% identity over the N-terminal 55 residues of the M5 protein, st4529 likely encodes a new serospecifically unique M protein.

These findings potentially affect vaccine development. Although new emm sequences were identified in a survey in the United States (5), the percentage of new strains with new emm sequences was much lower (6%) than was found with these Malaysian isolates, emm typing of a larger number of strains from rheumatic fever-and rheumatic heart disease-endemic areas is required to deduce amino acid sequences for the development of a suitable M protein-based vaccine.

Acknowledgments

We thank Sukeri Kasni and Theresa Hoenes for technical assistance.

This work was supported by University Kebangsaan Malaysia sabbatical leave grant and Fulbright Fellowship Award (1997) to Farida Jamal.

References

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(2.) Musser JM, Kapur V, Kanjilal S, et al. Geographical and temporal distribution and molecular characterization of highly pathogenic clones of Streptococcus pyogenes expressing allelic variants of pyogenic exotoxin A (scarlet fever toxin). J Infect Dis 1993; 167:337-46.

(3.) Kaplan EL. Global assessment of rheumatic fever and rheumatic heart disease at the close of the century. Circulation 1993;88:1964-72.

(4.) Beall B, Facklam R, Thompson T. Screening emm-specific PCR products for routine and accurate typing of group A streptococci. J Clin Microbiol 1996;34:953-8.

(5.) Beall B, Facklam R, Hoenes T, Schwartz B. Survey of emm gene sequences and T-antigen types from systemic Streptococcus pyogenes infection isolates collected in San Francisco, CA. Atlanta, GA and Connecticut in 1994 and 1995. J Clin Microbiol 1997;35:1231-5.

(6.) Jamal F, Pit S, Johnson DR, Kaplan EL. Characterization of group A streptococci isolated in Kuala Lumpur. J Trop Med Hyg 1995;98:343-6.

(7.) Johnson DR, Sramsk J, Kaplan EL, Bicova R, Havlicek J, Havlickova H, et al. Laboratory diagnosis of group A streptococcal infection. Geneva: World Health Organization; 1996.

Farida Jamal,(*) Sabiha Pit,(*) Richard Facklam,([dagger]) and Bernard Beall([dagger])

(*) University Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, Kuala Lumpur, Malaysia; and ([dagger]) Centers for Disease Control and Prevention, Atlanta, Georgia, USA