Emerging Infectious Diseases
[Volume 5 No.2 / March - April 1999]

Synopses

emm Typing and Validation of Provisional M Types for Group A Streptococci (ft 1)

R. Facklam,* B. Beall,* A. Efstratiou, V. Fischetti, D. Johnson, E.
Kaplan, P. Kriz, M. Lovgren,# D. Martin,** B. Schwartz,* A. Totolian,
D. Bessen, S. Hollingshead, F. Rubin, J. Scott,## and G. Tyrrell
*Centers for Disease Control and Prevention, Atlanta, Georgia, USA;
Central Public Health Laboratory, London, United Kingdom; The Rockefeller
University, New York, New York, USA; University of Minnesota, Minneapolis,
Minnesota, USA; National Institute of Public Health, Prague, Czech
Republic, #National Center for Streptococcus, Edmonton, Alberta, Canada;
**Communicable Disease Group, ESR, Porirua, New Zealand; Institute of
Experimental Medicine, St. Petersburg, Russia; Yale University, New
Haven, Connecticut, USA; University of Alabama, Birmingham, Alabama, USA;
National Institutes of Health, Bethesda, Maryland, USA; and ##Emory
University, Atlanta, Georgia, USA

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      This report discusses the following issues related to typing of
      group A streptococci (GAS): The development and use of the 5'
      emm variable region sequencing (emm typing) in relation to the
      existing serologic typing system; the designation of emm types
      in relation to M types; a system for validation of new emm
      types; criteria for validation of provisional M types to new
      M-types; a list of reference type cultures for each of the
      M-type or emm-type strains of GAS; the results of the first
      culture exchange program for a quality control testing system
      among the national and World Health Organization collaborating
      centers for streptococci; and dissemination of new approaches
      to typing of GAS to the international streptococcal community.

The Lancefield M-typing system, a typical serologic system based on
antigen-antibody reactions, is dependent on the preparation of
type-specific antisera and extraction of a protein identified as M protein
on the surface of group A streptococci (GAS) (1). The antisera against the
M-protein antigens are produced with whole-cell streptococcal vaccines used
to immunize rabbits. Acceptable antisera contain specific-precipitin
antibodies and type-specific antibodies that must enhance the phagocytosis
of the strain used to immunize the rabbit (2,3). The precipitin antibodies
are made specific by absorption of the serum with streptococcal cells to
remove the carbohydrate group antibodies and any cross-reactive precipitin
antibody to heterologous M-type strains. Each rabbit antiserum is tested
for reaction with antigens of all known M types.

Approximately half of GAS strains produce an apoproteinase, an enzyme that
causes mammalian serum to increase in opacity. This reaction is called the
serum opacity factor reaction, and the responsible enzyme is referred to as
opacity factor (OF). The OF enzymes are OF type-specific because each M
type that produces OF can induce type-specific OF antibodies that can be
used in OF inhibition tests (3,4). Preparation of the OF antisera and
specific details of the OF tests are described elsewhere (3,4). Some
laboratories have used OF typing to predict M types in epidemiologic
investigations. Even though it is not uniformly agreed that OF typing
antisera results should be reported as M or provisional M-typing results,
anti-OF antisera in many situations predict the M type in a type-specific
manner. For reporting purposes, if cultures are identified with M- or
OF-typing antisera, they should be identified as M type or OF type.

Reference strains used to prepare the M antisera (several of which were
originally described by Griffith in 1935) were historically obtained from
the Lancefield collection, Rockefeller University, New York; however more
recently, reference strains have been available from other reference
laboratories. M-types 1 through 51 were designated in the laboratory of Dr.
Rebecca Lancefield between 1928 and 1945. M-types 52 through 81 were
submitted by various investigators to reference laboratories in Atlanta,
London, New York, and Prague between 1965 and 1976 for confirmation. Some
laboratories believe that certain strains in the Lancefield collection do
not adequately express M protein and are unsuitable for the production of
M-type antiserum. Preparing M-type antiserum or a typing system accurately
related to the Lancefield system requires documented reference strains from
one of the internationally recognized reference laboratories. (ft 2)

In the Lancefield typing system, strains representing types 7, 16, 20, and
21 (originally described as Griffith) are not GAS but belong to groups C
and G. M-type 10 is the same serotype as M12, M-type 24 is the same as M45,
and M-type 35 is the same as M49; thus, designations of serotypes 7, 10,
16, 20, 21, 35, and 45 are not included in the Lancefield M-typing system 1
to 81 for GAS.

The emm gene of S. pyogenes is the gene that encodes the M protein. The M
protein, responsible for the bacterium's capacity to resist phagocytosis,
is a major virulence factor in GAS. The 5' ends of emm genes are highly
heterogeneous and encode for the serotype specificity used for the M-typing
system developed by Dr. Lancefield in 1928 (1). Producing type-specific
M-typing antisera is difficult and specialized; no attempt has ever been
made to produce them commercially, and only a few international reference
laboratories prepare them. The resurgence of rheumatic fever cases in the
United States and the emergence of severe infections (streptococcal toxic
shock and necrotizing fasciitis) caused by GAS in the 1980s and 1990s
indicated the need to reassess typing strategies for GAS. Since production
of M-type precipitating antisera is very expensive and labor-intensive, the
potential usefulness of a nonserologic typing system for GAS sequencing the
5' end of the M protein (emm) gene toward a molecular-based typing system
was examined.

emm Typing System for GAS

Before an emm genotype-based typing scheme was developed for GAS, the
nucleotide sequence at the 5' ends of emm genes had been reported for many
strains representing M-types 1-81 and several provisional M types (PT)
(5,6). However, it was not always evident that true reference strains had
been used. The knowledge gained from these studies provided the impetus for
exploring the feasibility of an emm-based genotyping system. Subsequent
studies based on sequencing the 5' emm genes from GAS reference strains and
clinical culture specimens have now been published (7,8). In all studies
involving emm sequence typing, 160 to 660 bases were sequenced from the 5'
terminal end of the emm gene. The methods of emm sequencing and emm gene
amplicon profiling restriction pattern techniques have been described
(7,8). Two isolates are regarded as sharing the same emm sequence type if
they are >/= 95% identical over their 5' end 160 nucleotides (includes
approximately 50 bp of the moderately conserved leader peptide-coding
region), allowing for one frame shift or in-frame insertion/deletion of no
more than seven codons (8). The results of emm gene sequencing of Dr.
Lancefield's reference strains types 1 to 51 and reference strains of
M-types 52 to 81, submitted to the Centers for Disease Control and
Prevention (CDC) Streptococcus Reference Laboratory as potential new M
types from 1967 to 1976 by various international investigators, are
summarized below.

The 5' end emm sequences of the following CDC M-type reference strains
matched the following sequences in GenBank that were submitted by other
investigators: Types 1, 2, 3, 4, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 19,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 33, 36, 37, 39, 41, 43, 44, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
72, 73, 74, 75, 76, 77, 78, 80, 81. The accession numbers for these and
many other emm sequences deposited in GenBank are listed in references 5-8
and at http://www.cdc.gov/ncidod/biotech/infotech_hp.html.

The 5' emm sequences from the CDC reference strains of GAS for the
following M types were submitted to GenBank: M-types 13 (AF025950), 32
(L47325), 34 (L47324), 38/40 (L46817), 42 (L46799), 67 (AF025949), 68
(AF025948), 69 (AF035838), 70 (AF035838), 71 (L46652). The 5' emm sequences
that were different from those submitted to GenBank by previous
investigators (6) are M-types 13, 67, 68, and 79. For M-type 13, the emm
sequence in GenBank was not from a recognized Lancefield typing strain; emm
sequence AF025950 should be considered the emm sequence for Lancefield
M-type 13. For M-types 67 and 68, the emm sequences in GenBank did not
match those from CDC. The reasons for this are unknown; however, emm
sequence AF025949 (M-type 67) and emm sequence AF025948 (M-type 68) were
obtained from the reference strains submitted to CDC by the investigators
who originally described these M types. For M-type 79, the emm sequence
obtained by the CDC investigators matched the emm sequence in GenBank
labeled M-type 80. Personal communication with the investigators who
submitted the emm sequences for M-types 79 and 80 indicated a transcription
error during submission of the sequences. Sequence U12004 is the correct
sequence for M-type 79. The emm sequences from the CDC reference strains
representing these four M types were confirmed by an independent
laboratory.

CDC has additional data on emm typing of more than 1,500 GAS isolates from
population-based studies, as well as random cultures from the United States
and several other countries. Nearly 100% of the cultures could be genotyped
by the emm typing system. In addition to determining the emm types of
reference strains of M-types 1 to 81, several provisional type strains as
well as new sequence type strains were typed by the emm typing procedure.
Only one of more than 1,500 GAS isolates could not be emm typed by current
methods. The emm sequence type of clinical isolates representing 35
distinct serotypes match the emm sequence type of the corresponding
reference strainincluded in this set of analyses are clinical isolates
that underwent M or OF serotyping at one of the internationally recognized
reference laboratories or elsewhere (8;9; B. Beall and D. Bessen, unpub.
data).

The historical correlations between T type, OF reaction, and M type are
largely unchanged when emm sequence type is substituted for M type
(3,4,7,8). Because this observation is based upon the analysis of >3,000
clinical isolates, the T type and OF reactions together constitute an
invaluable second-tier method for further confirmation of the grouping of
closely related isolates, as well as for resolution of unrelated sets of
organisms (R. Beall and R. Facklam, unpub. data).

When comparing a phenotypic-based typing scheme to a genotypic-based
scheme, complete concordancy is not expected. Although for most types the M
serotype is paired with a unique emm sequence type, there are several
discrepancies. In five well-documented instances, 5' end emm sequences are
>95% identical for two distinct M serotypes: M-types 27L and 77, 38 and 40,
44 and 61, 50 and 62, and 65 and 69 (10). The molecular basis for a lack of
concordancy can differ for each unique M-type/emm-type pair. A few critical
nucleotide substitutions at the 5' emm region could result in new, dominant
antigenic epitopes that lead to the generation of a distinct serologic
type. Alternatively, emm genes can occasionally undergo horizontal exchange
and move onto a new genetic background, as appears to be the case for emm44
and emm61 (6); in this instance, additional dominant, polymorphic antigens
may exist that can also be detected by M-typing sera. For the five examples
cited above, introduction of a second typing scheme (T-agglutination and OF
reaction) has proven useful in distinguishing between pairs displaying
identical emm sequence types, but distinct M serotypes.

The discriminatory power of the genotypic emm sequence-typing scheme
approximates that of the phenotypic M-serotyping scheme. For most M
serotypes, there is a one-for-one relationship with a unique emm sequence
type. The selection of 95% sequence identity as the cutoff value for
defining the emm sequence type is based on empirical measures that best
match the level of resolution achieved by M serotyping. In several
examples, the emm sequence of one emm-type/M-type pair displays a
relatively high level of sequence identity (but <95%) to a second, unique
emm-type/M-type pair. For example, the emm3 and emm31 sequences share 91.3%
identity over their first 160 bases; emm2 and emm73 are 89% identical over
their first 160 bases of 5' end sequence, and this similarity is increased
to 92.3% identity over their first 326 5' bases. Conceivably, certain
genetic changes, such as a single bp insertion in the emm hypervariable
region followed by a single bp deletion much farther downstream, could
alter the reading frame of the gene and hence the antigenic structure and
serotype of the emm gene product; however, this kind of variant has rarely
been encountered in CDC surveys. Other genetic changes, such as synonymous
substitutions, have no effect on phenotype. In some instances a single
deletion or insertion of seven or fewer codons within the hypervariable 5'
end 160 bp had no effect on the predicted M serotype (B. Beall, unpub.
data). Therefore, an emm gene with less than 95% sequence identity to other
emm genes may confer a new M serotype specificity. Thus, two isolates are
regarded as sharing the same emm sequence type if they are >/= 95% identical
over their first 160 nucleotides, allowing for one frame shift or in-frame
insertion/deletion of no more than seven codons (8). The 95% identity
cutoff is not expected to match perfectly what can be achieved by serologic
methods.

Most of the GAS isolates that are deemed nontypable by serologic methods
can be genotyped through emm sequence determination. Furthermore, the M
serotype does not always match the emm sequence-type (9,11). However, among
the hundreds of strains analyzed by the streptococcal reference laboratory
at CDC no discrepancies were observed between M serotype and emm sequence
type. The full extent of putative M-serotype/emm sequence-type
discordancies is not known, and the explanations for such a lack of
congruency are numerous. A more complete understanding of the basis for
discrepancies will be forthcoming as the emm sequence-typing method becomes
more widely implemented.

The emm-typing system is a useful and reliable epidemiologic tool for
subdividing GAS. Because it is independent of emm gene expression and can
often discriminate between biologically distinct isolates that may be only
weakly antigenic or nontypeable, emm sequence typing has the potential to
classify isolates that have been difficult to type by serologic methods.

Designation of M, Provisional M, and emm Sequence Types

GAS strains fall into three categories: Validated M types, provisional M
types, and emm sequence types. If a laboratory has prepared antiserum to an
unknown strain and the serum has type-specific precipitating antibodies, as
well as bactericidal antibodies directed to that strain, verified by one of
the six original reference laboratories, an M-type designation can be
assigned to that strain. If two laboratories (at least one being one of the
six reference laboratories) produces type-specific precipitating and
bactericidal antiserum to the strain, the strain also qualifies as a new M
type.

When a laboratory has prepared antiserum as described above to an unknown
strain but the specificity of this antiserum has not yet been confirmed by
a second reference laboratory, that strain is designated a provisional
type. The requirements for conventional validation of new M types will be
described elsewhere. A third category are sequence types or emm types,
which are strains typed by sequencing the emm gene. If cultures are
identified by emm sequencing, they should be reported as emm type.

Validation Procedures and Nomenclature of New emm Sequence Type Strains

Published emm sequences from studies conducted in New Zealand and Australia
included several new emm sequences included in GenBank (12,13). In
addition, data from the CDC studies (B. Beall and R. Facklam,
http://www.cdc.gov/ncidod/biotech/infotech_hp.html) indicated that more
than 30 unknown emm sequences were identified among the 1,500 isolates of
GAS that had been emm sequenced. A working group of representatives of each
of the six international reference centers in Canada, New Zealand, Czech
Republic, United Kingdom, and United States, was charged with establishing
a definitive protocol both for submission of new emm sequences and for
subsequent validation of new emm types. As an interim measure, unique 5'
end emm sequences proposed as new emm types must be confirmed by a second
laboratory; at least one of the two laboratories should be the
streptococcal reference laboratory at CDC. If the uniqueness of the emm
sequence can be confirmed by the second laboratory, the original
investigator or one of the confirming laboratories will submit the findings
to the Working Group, which will determine whether the strain should be
assigned a new emm type number (e.g., emm94). In addition to sequence
uniqueness, additional factors may be considered by the working group when
making this decision; for example, previous requirements for assigning
regular M- and provisional M-type numbers to strains were restricted to
strains of particular clinical significance or to those occurring in a
population "with significant frequency." Another remaining unresolved issue
is whether or not all new emm reference strains should actively express M
protein. A lack of surface expression of this emm gene product will
preclude any possibility for correlation of emm type with classic serologic
type or subsequent evaluation of biological significance. The relationship
of emm sequence to biological function needs to be further explored.

CDC has validated six emm sequences submitted to GenBank by Australian
investigators (12-14) and one emm sequence from a strain submitted to CDC
by an investigator in the United States (15); these sequences should all be
considered for official status as new emm types. Four additional isolates
were examined at the CDC laboratory for which emm sequences had been
submitted to GenBank (12-14). Strains STBSB75, ST1293, ST87/156, and STNS27
were shown to have the same emm types as M-type 70, M-type 76, PT2110, and
PT5757, respectively. Therefore, these four emm sequences should not be
accepted as new emm types, and their sequences should be reidentified in
GenBank.

CDC has identified 10 new emm types from population-based studies of GAS
invasive disease (7,8, unpub. data). In addition, eight new emm sequences
from Brazil, six from Malaysia, three from Papua New Guinea, three from
India, two from Ethiopia, two from Gambia, and one each from New Zealand
and Chile have been confirmed by a second laboratory for emm sequence
uniqueness, for 36 potentially new emm types.

List of Reference Strains

The WHO Collaborating Laboratory for Reference and Research on Streptococci
in Prague has prepared a database of the reference type strains to be used
for research and antiserum production from information provided by the six
international Reference Centers in Canada, New Zealand, Czech Republic,
United Kingdom, and United States. Although all the reference strains on
the list at one time had demonstrated survival in the in vitro bactericidal
test (presumably reflecting functional M protein), the reference strains
should be retested for survival in the bactericidal test before use in
research. Dr. Lancefield's strains types 1 to 50 are listed at
http://www.rockefeller.edu/vaf/

Because listing of type strains may be slightly different for each culture
collection, when cultures are obtained, the strains should be properly
identified. The strains for reference types 1 to 50 should be traced to the
Lancefield collection, from which the M-typing system was derived. In the
past, if a reference strain lost the capacity to express M protein on the
bacterial cell surface, that strain was passaged in vivo and selected for
the increased presence of M protein; therefore, many derivatives of the
original reference cultures are in use by the reference laboratories, and
none are known to have undergone change in their emm gene nucleotide
sequence. The American Type Culture Collection (ATCC) has Lancefield's
strains 1 to 50, the UK National Collection of Type Cultures has types 1 to
81, as well as the provisional types, and the Czech Culture Collection in
Prague also has types 1 to 81 on deposit. Reference cultures 51 to 81 have
been deposited in the ATCC by the CDC investigators who will also deposit
the provisional type strains shortly. Plans are to continue the deposition
of cultures of new emm types as they are confirmed.

Additionally, the CDC Streptococcus Laboratory has established an emm-type
database at http://www.cdc.gov/ncidod/biotech/infotech_hp.html for use as a
sequence comparison tool and additional documentation of all of the
verified M and emm types. The database contains detailed information on the
CDC method of emm typing and provides additional background on each
reference strain and most known emm sequence types. Sequence comparisons
can be done by using BLAST (Basic Local Alignment Search Tool). The data
are prereviewed for errors and discrepancies, and all newly deposited
emm-types are first verified directly by the CDC reference laboratory. At
this time, until a complete validation protocol is established,
investigators who discover new emm sequence types should submit their
isolates to the CDC Streptococcus Laboratory to confirm uniqueness. As a
first step, investigators should search the CDC database for emm sequences;
Genbank can be used as a second step, since it is always possible that
neither database will necessarily have all known emm sequences of GAS at
any given time. Furthermore, the CDC database will include the accepted emm
type designation for those types, whereas a possible incorrect emm sequence
may have been submitted to GenBank by an investigator.

Validation of Provisional M Types to New M and emm Types

The Table lists the provisional M-type strains and the status of
validation. Collaborative investigations involving the six reference
laboratories included several other provisional M-type strains and has
confirmed the following: PT179 fulfills all the phenotypic criteria for an
M type; however, only anti-OF serum has been prepared. Therefore, the
status of this strain as a potential new M type remains on hold until
further supporting data can be provided. PT4854, Colindale Laboratory,
United Kingdom (UK), had a closely matching emm type as M-type 43; this
finding correlated to serologic tests at CDC and UK laboratories as M-type
43 that demonstrate that both strains reacted with M43 typing antiserum.
PT3800, Prague, Czech Republic (CZ), has the same emm type as M-type 65,
which correlated to serologic tests performed at the National Streptococcus
Laboratory in Edmonton, Canada, showing that both strains reacted with M65
typing antiserum. PTYE327 (CZ) has the same emm type as PT2841(UK), which
correlates to serologic tests performed in both the Colindale and Prague
laboratories. PT1437, Porirua Laboratory, New Zealand (NZ), has the same
emm type as PT4245 (UK), which correlates to serologic tests performed in
both the UK and NZ laboratories. ST2974.95 (CDC) has the same emm type as
PT5118 (NZ). ST2974.95 was shown to be PT5118 in serologic tests performed
in the Porirua Laboratory. In summary, PT4854, PT3800, PTYE327, PT1437, and
ST2974.95 should be identified as emm43 (M43), emm65 (M65), emm87 (M87),
emm89 (M89), and emm92, (M92) respectively. The sources of most provisional
types were first documented in 1985 (16).

 Table. Recommended action for M and emm                     The following
 designation for provisional M types(sup a)                  emm types were
                                                             isolated from
 ----------------------------------------------------------- patients with
 Provisional                      CDC ID        New emm      severe
 type                ID number    number       or M type     invasive
                                                             disease in the
 ----------------------------------------------------------- United States;
 PT180(UK)(sup b)    NCTC12062    SS-1395          82        emm82, emm83,
                                                             emm86, emm87,
 PT2110(UK)          NCTC12064    SS-1400          83        emm88, emm89,
 PT2233(UK)          R75/2233     SS-1449          84        and emm92.
 PT2612(UK)          R76/2612     SS-1447          85        These isolates
                                                             comprised
 PT2631(UK)          R76/2631     SS-1448          86        12.7% of all
 PT2841(UK)          NCTC12065    SS-1399          87        isolates
 PT3875(UK)          R67/3875     SS-1455          88        identified
                                                             during these
 PT4245(UK)          NCTC12067    SS-1397          89        studies. Types
 PT4931(UK)          NCTC12068    SS-1396          90        emm82, emm85,
                                                             emm87, and
 PT5757(UK)          NCTC12056    SS-1398          91        emm89 were
 PT5118(NZ)                       SS-1460          92        associated
 PTPotter41(UK)      R76/2631     SS-1493          93        with epidemic
                                                             investigations
 ----------------------------------------------------------- in the United
 (sup a)For definitions of M and emm designations, see       States. Many
 section in text, Designation of M, Provisional M, and emm   of these new
 Sequence Types.                                             emm types were
 (sup b)Abbreviations; UK= United Kingdom, NZ= New Zealand.  also
                                                             identified
                                                             from other
countries, including Argentina, Brazil, Bulgaria, Chile, Colombia, Denmark,
India, Korea, Malaysia, New Guinea, and Poland. Other reference
laboratories have reported these new M types (by using provisional type
identifiers) in a variety of infections, including rheumatic fever and
acute glomerulonephritis (17,18).

An External Quality Assurance Typing Program among International Reference
Laboratories

At the request of the World Health Organization (19), a quality assurance
program on GAS typing has been established by the Central Public Health
Laboratory, London, United Kingdom. Ten GAS isolates were examined by six
different laboratories, and the results of the first distribution showed a
very good correlation between laboratories. Emm typing by one laboratory
correlated very well with M typing by the other five laboratories. Only
minor differences were noted among the T- and OF-typing results; no errors
were found among the M-typing results. Responsibility for sending 10
cultures to the other five centers twice a year would be rotated among the
six reference centers and a report of the quality assurance program will be
presented at the next International Lancefield Society meeting in New
Zealand in the fall of 1999.

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(ft 1)Information in this report was first presented at an International
Workshop on Demonstration of emm Typing and Validation of Provisional M
types for Group A Streptococci held at the Centers for Disease Control and
Prevention, Atlanta, Georgia, August 26-28, 1997.

(ft 2)Centers for Disease Control and Prevention, Atlanta, Georgia, USA;
Central Public Health Laboratory, London, United Kingdom; The Rockefeller
University, New York, New York, USA; University of Minnesota, Minneapolis,
Minnesota, USA; National Institute of Public Health, Prague, Czech
Republic, National Center for Streptococcus, Edmonton, Alberta, Canada;
Communicable Disease Group, ESR, Porirua, New Zealand.

  Dr. Richard Facklam is chief of the Streptococcus Laboratory, Division of
Bacterial and Mycotic Diseases Division, National Center for Infectious
Diseases, CDC. His major fields of interest include improvement in
laboratory procedures for the diagnosis of acute respiratory tract
infections, taxonomy of streptococci and related gram-positive cocci,
identification of virulence factors associated with bacterial respiratory
pathogens, and development of new systems for epidemiologic study of the
transmission of bacterial respiratory pathogens.

      Address for correspondence: Richard R. Facklam, Centers for Disease
Control and Prevention, 1600 Clifton Road, Mail Stop C02, Atlanta, GA
30333, USA; fax: 404-639-1379; e-mail: rrf2@cdc.gov.

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Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

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