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177
A Genetic-Based Evaluation of the Principal Tissue Reservoir for Group A
Streptococci Isolated from Normally Sterile Sites
Therese R. Fiorentino, Bernard Beall, Patricia Mshar,
and Debra E. Bessen
Department of Epidemiology and Public Health, Yale University School
of Medicine, New Haven, and State of Connecticut Department of
Public Health, Hartford, Connecticut; Childhood and Respiratory
Diseases Branch, Centers for Disease Control and Prevention,
Atlanta, Georgia
The primary sites of infection and principal reservoirs for transmission of group A streptococci
are the nasopharyngeal mucosa and the impetigo lesion. However, pharyngitis and impetigo are
rarely observed prior to invasive disease, and, thus, the origin of invasive strains is largely unknown.
As part of an active surveillance program, group A streptococci were obtained from normally sterile
tissue sites of Connecticut residents during a 6-month period. Organisms were analyzed for genetic
markers that distinguish between strains that use the nasopharynx versus an impetiginous lesion
as their primary site for infection. The nasopharyngeal marker was observed for most sterile-site
isolates, suggesting that the upper respiratory tract is the principal reservoir from which organisms
causing invasive disease are disseminated. Genotypic analyses of sterile-site isolates support the
view that additional factors, aside from a recent emergence of a few virulent clones, are important
contributors to invasive group A streptococcal disease.
The overall incidence of severe invasive group A streptococcal infection has likely undergone a dramatic increase in several
parts of the world since the mid-1980s [1 – 12]. Group A streptococci are among the most common bacterial pathogens afflicting humans, and disease usually takes the form of a non –
life-threatening and self-limiting pharyngitis or superficial impetigo. The nasopharyngeal mucosa and impetiginous lesion
of the human host are the two principal tissue reservoirs for
the maintenance and transmission of group A streptococci.
However, in invasive group A streptococcal disease, the portal
of entry into the host is often not known [2, 4, 8, 9, 11].
The concept of distinct groups of throat and skin (i.e., impetigo) strains of group A streptococci is well recognized [13 –
15]. A recent study [16] showed that populations of nasopharyngeal- and impetigo-derived group A streptococci are genetically distinct in the portion of the chromosome (emm gene
region) that encodes for the determinants of a widely used
serologic typing scheme (M proteins). Although ú80 different
M serotypes have been identified, the arrangement of emm and
Received 7 November 1996; revised 6 February 1997.
Presented in part: XIII Lancefield International Symposium on Streptococci
and Streptococcal Diseases, Paris, September 1996 (abstract L26).
Human experimentation guidelines of the state of Connecticut were followed
in the conduct of clinical research.
Financial support: CDC – State of Connecticut Department of Public Health
Emerging Infections Program, American Heart Association (D.E.B. is an Established Investigator), Donaghue Medical Research Foundation, and National
Institutes of Health (AI-28944).
Reprints or correspondence: Dr. Debra Bessen, Yale University School of
Medicine, 333 Cedar St., Box 208034, Dept. of Epidemiology and Public
Health, New Haven, CT 06520.
The Journal of Infectious Diseases 1997;176:177–82
q 1997 by The University of Chicago. All rights reserved.
0022–1899/97/7601–0022$02.00
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emm-like genes on the chromosome takes the form of only five
major patterns [17, 18]. The objective of this study is to use
the tissue-related emm gene patterns as markers for predicting
the principal reservoir for group A streptococcal strains isolated
from normally sterile tissue sites.
Materials and Methods
Case reporting and collection of bacterial isolates. Connecticut is one of four states participating in an Emerging Infections
Program (EIP) through a cooperative agreement with the Centers
for Disease Control and Prevention (CDC). One of the projects of
the program involves active population-based laboratory surveillance for invasive disease caused by several bacterial pathogens,
including group A streptococci. In January 1995, invasive group
A streptococcal disease was made a reportable disease in the state
of Connecticut. As a result, hospital laboratories were required to
send group A streptococcal isolates obtained from normally sterile
tissue sites to the State of Connecticut Department of Public
Health. In March 1995, active laboratory surveillance for invasive
group A streptococcal disease was implemented in all 35 acutecare hospital laboratories in Connecticut. On a monthly basis, hospital microbiologists completed a form listing all patients having
invasive group A streptococcal disease. If a report form was not
received, the hospital microbiologist was contacted by telephone
for case information. Clinical information was obtained from the
patient’s medical record. Diagnoses of streptococcal toxic shock
syndrome (Strep TSS) met the case definition established by the
CDC [19].
During the 6-month survey period for this study (1 March
through 31 August 1995), group A streptococcal isolates obtained
from sterile sites in 64 Connecticut state residents were sent to the
State of Connecticut Department of Public Health, and all 64 isolates are included in this report. Sterile-site isolates were obtained
from blood, normally sterile tissue fluids (e.g., synovial, pleural),
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Fiorentino et al.
Figure 1. Chromosomal arrangement of emm and emm-like genes.
emm and emm-like genes fall into 1 of 4 major subfamilies (SF)
defined by nucleotide sequence differences in portion of emm or emmlike gene encoding peptidoglycan-spanning domain [17]. ú99% of
group A streptococcal strains examined have 1, 2, or 3 distinct emm
or emm-like genes arranged in 1 of 5 chromosomal patterns (A – E)
shown. Nomenclature shown for each of 3 emm and emm-like gene
positions (mrp, emm, enn) within emm chromosomal region is that
proposed by Whatmore et al. [21].
or surgical tissue biopsies. Isolates were collected from throughout
the state; the three most populous counties each accounted for
25%–29% of the isolates, and the remaining five counties shared
17% of the total. A retrospective audit of hospital microbiology
laboratories uncovered 12 additional cases of group A streptococcal invasive disease. In addition, isolates derived from normally
sterile tissue sites of 9 patients were never sent to the state laboratory; since these 21 bacterial isolates were not available for molecular analyses, those cases were excluded from the study.
T antigen serologic typing was performed by standard methods.
Strains associated with recent cases of severe invasive disease
from elsewhere in the United States (i.e., strains with an MGAS
designation), which were previously evaluated by multilocus enzyme electrophoresis [20], were provided by James Musser (Baylor
University, Houston).
Determination of emm chromosomal patterns. The relative arrangement of emm genes on the chromosome was determined as
previously described [16–18] (figure 1). Chromosomal DNA was
prepared from overnight cultures of group A streptococci and used
as a template for a series of polymerase chain reaction (PCR)
amplifications. Subfamily-specific primers were paired with a second primer capable of hybridizing to DNA within the emm chromosomal region of at least some strains, and a series of overlapping
PCR amplifications were performed using various primer pair combinations. Agarose gel electrophoresis was used to determine the
presence or absence and molecular size of reaction products, and
a linear map of the emm chromosomal region was constructed
based on the data [17, 18]. The arrangement of emm genes within
each bacterial strain was scored as pattern A, B, or C, pattern D,
or pattern E (figure 1). PCR amplifications included Taq DNA
polymerase (Promega, Madison, WI, or Boehringer Mannheim,
Indianapolis), the manufacturer’s buffer (containing 1.0 mM
MgCl2), and 0.8 mM of each primer. Thirty cycles were performed,
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JID 1997;176 (July)
with an anneal temperature of 557C and an extension time of 1–
3 min, depending on the sizes of the expected products.
Arbitrary-primed PCR, or random-amplified polymorphic DNA
(RAPD). Chromosomal DNA was purified and PCR amplifications were done as described above, with the following modification: A single 10-mer oligonucleotide primer (p17; 5*-GATCTGACAC-3*) was used at a concentration of 2.0 mM and MgCl2
was included at 2.5 mM [22]. Thermal cycling was performed as
described [22]: Cycles 1–5 were run at 947C for 30 s, 377C for 2
min, and 727C for 5 min, followed by cycles 6–40 at 947C for 30
s, 377C for 1 min, and 727C for 1.5 min. Gels containing 1.0% ME
agarose (FMC BioProducts, Rockland, ME) were used to separate
reaction products. For all DNA templates that initially yielded
only a few or no bands, template DNA was tested at reduced
concentrations; for all strains, complex and reproducible banding
patterns were obtained. Each DNA template was tested for PCR
amplification a minimum of three times; experimental repeats
helped to reduce any ambiguities arising from variations in the
intensity of certain bands. Each RAPD profile is represented by
isolates that are identical in bands migrating between 0.4 and 2.4
kb. All RAPD analyses were completed at Yale University without
prior knowledge of the emm nucleotide sequence and T antigen
serotyping data, which were determined at CDC.
Detection of the speA gene. PCR amplification of chromosomal DNA was done using primers 5*-ATGGAAAACAATAAAAAAGTATTG-3* (forward) and 5*-TTACTTGGTTGTTAGGTAGACTTC-3* (reverse), corresponding to amino acids 1–8 and 245
through stop codon, respectively, of the published speA sequence
[23]. Conditions used for PCR amplification for speA were the
same as those used for emm chromosomal mapping.
Emm gene sequencing. Nucleotide sequence determinations
were made for the 5* ends of emm genes as previously described
[24], except that primer emmseq2 (5*-TATTCGCTTAGAAAATTAAAACAGG-3*) was used at an annealing temperature of
557C. For all isolates, including those with multiple emm genes, the
gene amplified with primers 1 and 2 [21] and used for nucleotide
sequencing is that designated ‘‘emm’’ in figure 1; the gene occupying the central position within the emm chromosomal region
most likely represents the gene encoding for the principal serologic
determinant (M type) [21, 25]. Sequences were designated as emm
types on the basis of identities or near identities to emm genes of the
standard M typing strains; the criterion was nucleotide sequence
identity §95% in the first 160 bases of the 5* end when compared
with Genbank sequences as defined in [24].
Results
Incidence of invasive disease. A total of 64 sterile-site isolates of group A streptococci were collected by the State of
Connecticut Department of Public Health between 1 March
and 31 August 1995. Each sterile-site isolate was obtained from
a different Connecticut resident. On the basis of 1990 census
data [26] and including the 21 additional cases for which no
organism was available for molecular analysis, the estimated
annual incidence of invasive group A streptococcal disease in
1995 was 5.17/100,000 residents.
Distribution of genetic markers for nasopharyngeal versus
impetigo reservoirs. In severe invasive disease, the portal of
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Tissue Reservoir of Invasive Streptococci
entry for the group A streptococcus is not always obvious.
We used the emm chromosomal patterns (figure 1) as genetic
markers for predicting the principal tissue reservoir for group
A streptococci isolated from normally sterile sites. In a previous
study, in which epidemiologically unrelated organisms derived
from only the nasopharyngeal mucosa or impetigo lesion were
evaluated, 96% of isolates displaying emm chromosomal patterns A, B, or C were derived from the nasopharyngeal mucosa,
whereas 86% of pattern D strains were isolated from impetigo
lesions [16]. The nasopharyngeal and impetigo isolates were
collected from throughout the world over a period of ú50
years.
In the Connecticut population-based study, õ2% of the sterile-site isolates exhibited emm chromosomal pattern D (table
1), a genetic marker for an impetigo reservoir. In striking contrast, 70% of the sterile-site isolates displayed emm chromosomal patterns A, B, or C, suggesting that at least two-thirds
of invasive infections were caused by strains that principally
reside in the throat. Pattern E strains, which as a group have
no clear-cut tissue site preference [16], constitute the remaining
28% of the Connecticut isolates. The overall distribution of
emm chromosomal patterns among the sterile-site isolates from
Connecticut closely paralleled the distribution observed for epidemiologically unrelated nasopharyngeal isolates and was significantly different (P õ .001, x2 analysis) from that exhibited
by impetigo-derived strains (figure 2).
Clinical correlates. emm chromosomal patterns of group
A streptococci were analyzed for correlations with both the
human tissue site from which each strain was isolated and
disease pathology. All isolates associated with Strep TSS or
necrotizing fasciitis (or both) displayed emm chromosomal patterns A – C (table 2). Thus, emm chromosomal patterns A – C
strains are associated with the more severe forms of group A
streptococcal invasive disease in the Connecticut population.
All of the Strep TSS – associated bacteria and 6 of the 7 necrotizing fasciitis – associated organisms had a detectable speA
gene. This result confirms previous studies demonstrating a
high correlation between the presence of speA and Strep TSS
[2, 20, 27, 28]. Overall, 33 of the isolates (52%) harbored the
bacteriophage-borne speA gene, and of these, all were represented by emm chromosomal patterns A – C (table 1). Blood
isolates comprised 43 (67%) of the 64 isolates, and no significant difference was noted between emm patterns A – C and
emm pattern E strains in terms of their association with bacteremia (table 2).
Extent of genetic diversity. The extent of diversity among
group A streptococci isolated from Connecticut residents was
ascertained by a combination of three methods (table 1): arbitrary-primed PCR (RAPD), nucleotide sequence determination
of the 5* portion of the central emm gene (defined in figure 1),
and serologic typing of the T antigen. It is important to note that
analysis of all RAPD profiles was completed at Yale University
without any prior knowledge of the data on emm gene nucleotide sequencing and T serotyping, which was performed at the
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179
Table 1. Genotypic analysis of 64 isolates of group A streptococci
derived from normally sterile tissue sites — Connecticut residents,
(March – August 1995).
RAPD* profile
emm patterns A – C
1‡
2
3
4§x
5
6
6Ø
7
8
9
10
11
emm pattern D
12
emm pattern E**
13††
14
15
15
15
16
17
18
19
20
21
22
23
24
25
26
Emm
gene
sequence†
T type
Presence or
absence of
speA
No. of
strains
represented
1
3
3
3
3
12
1
6
3
5
ND
18
1
3
3
3
3
11/12
1
6
NT
NT
ND
NT
/
/
/
/
0
0
0
/
0
0
0
/
18
3
2
3
4
4
1
6
1
1
1
1
80
14
0
1
28
28
11
11/12
13
13
3/13/B
13
11
11
25
25
3
2
4
14
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
28
28
pt4245
pt4245
77
13
13
73
11
78
75
75
st2346‡‡
2
4
59
NOTE. RAPD Å random amplified polymorphic DNA; NT Å not typeable; ND Å not determined.
* Profiles 1 – 26 differ by §1 bands that migrate between 0.4- and 2.4-kb
molecular size markers.
†
All emm1 isolates display sequence identities of §99.5% to Genbank
accession no. U11940 over 5* end 231-base overlap, with 1 exception wherein
3-base deletion is noted. Similarly, all emm3 isolates display sequence identities
of §99% to Genbank accession no. U11945 over 5* end 243-base overlap.
‡
Two 18 RAPD1 isolates were not evaluated for either emm type or T type.
§
One strain from each of RAPD4 and RAPD5 groups was not evaluated
for emm or T type.
x
Two distinct genotypes were derived from 1 of RAPD4 ‘‘isolates.’’ Second
genotype, which displays unique RAPD profile that was not reported, has emm
chromosomal pattern that is consistent with data collected at CDC (emm11,
T11/12, opacity factor positive). Thus, there is uncertainty as to which genotype
caused invasive disease.
Ø
Isolate is genotypically distinct from RAPD6-emm12-T11/12 isolates according to additional polymerase chain reaction (PCR) analyses of emm chromosomal region (unpublished findings).
** All emm pattern E isolates exhibited opacity factor activity.
††
RAPD13 and RAPD14 profiles differ at 2 major bands; however, there
are commonalities between RAPD13 and RAPD14 strains in PCR amplification
products corresponding to portions of emm chromosomal region.
‡‡
st2346 represents emm type not previously encountered; closest Genbank match is emmpt4245, having 79.4% identity over 286 bp of 5* emm
sequence.
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Fiorentino et al.
JID 1997;176 (July)
Figure 2. Distribution of emm chromosomal patterns
of sterile-site isolates compared with distribution for
strains derived from nasopharynx or impetigo lesion.
Sterile-site isolates of group A streptococci (n Å 64) were
obtained from Connecticut residents during March – August 1995. Comparison data shown for nasopharyngeal
and impetigo isolates were previously reported [16]; vast
majority of throat isolates came from cases of uncomplicated pharyngitis or acute rheumatic fever, and impetigo
lesions from which bacteria were isolated were typically
superficial rather than having deep tissue involvement.
CDC. Collectively, the 64 isolates exhibited 26 distinct RAPD
profiles. There were 21 unique combinations of emm gene
sequence type and T serotype among the 58 strains that were
subjected to those evaluations.
The RAPD profile representing the most isolates was RAPD1
(n Å 18), and all RAPD1 organisms exhibited emm1/T1. Three
previously characterized electrophoretic type (ET) 1 isolates
[20] displayed the RAPD1 profile (data not shown); ET1 and
ET2 represent 2 clonal groups that likely account for many of
the invasive group A streptococci isolated within the past decade [2 – 5, 8 – 10, 20, 29, 30]. Four RAPD profiles (RAPD2 –
RAPD5, n Å 12) corresponded with emm3/T3; two of these
profiles (RAPD2 and RAPD3) were identical to the RAPD
profiles of ET2 strains MGAS157 and MGAS158, respectively.
The RAPD2, RAPD4, and RAPD5 profiles each differed from
RAPD3 by one or two bands. Since two distinct RAPD profiles
fell into a singular ET, and all four of the RAPD2 – RAPD5
Table 2. Clinical correlates of emm chromosomal patterns of group
A streptococci isolated from Connecticut residents.
No. of isolates associated with each disease
Emm chromosomal pattern*
Disease
A–C
Streptococcal toxic shock
syndrome
Necrotizing fasciitis
Bacteremia without focus
Cellulitis
Bacteremia (total)
None of the above†
5
7
11
15
31
6
(11)
(16)
(24)
(33)
(69)
(13)
D
0
0
0
1
1
0
(0)
(0)
(0)
(100)
(100)
(0)
E
0
0
5
4
11
5
(0)
(0)
(28)
(22)
(61)
(28)
Total
5
7
16
20
43
11
NOTE. Nos. in parentheses represent % of isolates within given emm
pattern group that are associated with each disease.
* Several isolates are associated with ú1 clinical manifestation. Of 64 isolates represented, 45 display emm patterns A – C, 1 displays pattern D, and 18
display pattern E.
†
Includes isolates associated with focal infections that were derived from
pleural fluid (n Å 3), synovial fluid or bursa (n Å 4), lymph node (n Å 1),
abscess (n Å 1), burn (n Å 1), muscle tissue (n Å 1).
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profiles shared the emm3/T3 type, we presume that isolates of
all four profiles are closely related. Thus, as many as 30 of the
64 sterile-site isolates (47%) from Connecticut are genotypically related to clones previously identified as causative agents
for a significant proportion of severe invasive group A streptococcal infections [3 – 5, 20]. However, equally important is
the finding that the remaining half of the sterile-site isolates
displayed extensive genetic diversity.
For the 58 strains that were analyzed by all three parameters
(RAPD, emm type, and T type), 28 unique combinations are
observed. Two RAPD profiles (RAPD1 and RAPD6) corresponded to emm1/T1, whereas four (RAPD2 – RAPD5) were
associated with emm3/T3. Two additional sets of strains with
identical emm type/T type combinations also had multiple
RAPD patterns (RAPD13 and RAPD14, RAPD21 and
RAPD22). The data are consistent with a previous study in
which RAPD analysis using the same oligonucleotide primer
(p17) had a higher discriminatory power than M protein serotyping [22].
In most instances, the strain contents of a given RAPD pattern were homogeneous with regard to the emm type/T type
combination. In all cases, every strain displaying a particular
RAPD profile was either positive or negative for the speA gene
(table 1). On the basis of this double-blind study, it appears
that RAPD analysis is a reliable tool for discrimination between
group A streptococci of distinct genotypes.
Discussion
The nasopharyngeal mucosa and impetigo lesion of the human host represent the two principal tissue sites for group A
streptococcal infection and transmission. This report compares
3 populations of group A streptococci that are derived from
different anatomic sites: normally sterile tissue, nasopharyngeal
mucosa, and impetiginous lesion. A comparison of the relative
distribution of emm chromosomal patterns among the 3 tissuedefined groups shows that the nasopharyngeal and sterile-site
isolates are strikingly similar, whereas the impetigo-derived
population is markedly distinct. If emm chromosomal pattern
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Tissue Reservoir of Invasive Streptococci
is a genetic marker for the principal tissue site for group A
streptococci, then one can conclude that the nasopharyngeal
mucosa is the principal reservoir for strains capable of causing
severe invasive disease in Connecticut. We assume that there
exists many individuals within the community that harbor in
their throats, without any severe ill effects, the same clones
that occasionally cause invasive disease [31]. From the throat
of a susceptible host, a given organism might undergo hematogenous spread or alternatively, it may gain access to normally
sterile tissue via secondary transfer through a break in the skin.
Although the relative distribution of emm chromosomal patterns was similar for the sterile site – and nasopharyngealderived populations, the 2 groups differed in a fundamental
way: The sterile-site isolates were epidemiologically related,
whereas the nasopharyngeal isolates were epidemiologically
unrelated. Coupled to this is the fact that the spectrum of M
types among the sterile-site versus nasopharyngeal groups is
significantly different [16]. Thus, the mode of sampling has no
obvious effect on the overall distribution of emm patterns. This
finding reinforces the view that there exists biologically important subpopulations of this bacterial species that are genetically distinct in the emm chromosomal region. Whether the
sterile-site isolates merely reflect the group A streptococcal
clones that are prevalent in Connecticut or whether they comprise a distinct subgroup of strains is not known.
Our findings are consistent with several other lines of evidence suggesting that group A streptococcal strains associated
with invasive disease reside principally in a throat reservoir
within the human population. A small percentage of patients
with invasive disease (typically õ20%) present clinically with
pharyngitis [2, 4, 8, 9, 11]. One can also argue that many of
the M types associated with invasive disease are typical of
‘‘throat’’ types [32], and that invasive disease occurs in communities at times when streptococcal impetigo is uncommon.
In a recent study on a single clone of group A streptococci
that was responsible for an outbreak of invasive disease in
Minnesota, the identical clone was found in association with
pharyngitis and asymptomatic throat carriage among other individuals within the same community [33]. The use of genetic
markers for predicting the principal tissue reservoir represents
a new approach that is well-suited for situations where more
direct evidence is difficult to obtain.
There is a wide spectrum of diseases having cutaneous
involvement that are caused by group A streptococci, including
impetigo, ecthyma, erysipelas, and cellulitis [34, 35]. Impetigo
is a superficial infection involving the epidermal layer of the
skin, and accompanying septicemia is rare. In some instances,
a mild untreated case of impetigo can progress to ecthyma,
causing lesions to penetrate deeper into the dermis. Cellulitis
can arise following a break in the skin, and the infection extends
to the subcutaneous tissue; from this site, the bacterium can
give rise to systemic infection by spread through the lymphatics
and bloodstream. It is generally accepted that group A streptococci giving rise to cellulitis can originate from either a naso-
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181
pharyngeal site or superficial impetigo lesion, whereby the organism is acquired via secondary transfer or from another
contact. In a previous study, the majority of isolates obtained
from cases of impetigo were derived from superficial lesions;
95% of the impetigo isolates analyzed display emm chromosomal patterns D or E [16].
In contrast to the impetigo-derived strains, 75% of the organisms obtained from Connecticut patients with cellulitis were
of emm chromosomal patterns A – C. Furthermore, of the 20
cases of cellulitis, blood isolates were obtained from 65% of
the patients. Thus, our findings are consistent with a common
clinical course for group A streptococcal cellulitis, whereby
bacteria are transferred from the throat to a break in the skin,
and deep cutaneous infection leads to bacteremia in some patients. The relatively high association of bacteremia with cellulitis in the Connecticut patients may reflect a unique virulence
property of emm1 and emm3 type isolates, which together represent 9 of the 13 bacteremic cases of cellulitis.
This prospective survey of invasive group A streptococcal
disease is distinct from many other population-based studies
in that the active surveillance system guarantees collection of
most sterile-site isolates from patients statewide. Furthermore,
the temporal and spatial limits are narrowly defined. The finding that almost half of the sterile-site isolates appear to be
genetically related to 2 clonal groups (represented by emm
types 1 and 3) underscores the contribution of a few virulent
clones to the disease burden. Equally striking is the finding
that more than half of the isolates display extensive genetic
diversity. The nonclonal nature of invasive group A streptococci is exemplified by the 18 emm pattern E isolates, which
are represented by 16 unique RAPD/emm type/T type combinations. Thus, many distinct clones have the capacity to cause
invasive disease [20, 36], and a wealth of genetic diversity is
displayed by this bacterial species within a relatively small
geographic region and over a short duration. Therefore, a recent
emergence of only a few virulent clones cannot fully account
for invasive group A streptococcal disease in the 1990s.
Acknowledgments
We thank Nancy Barrett for assistance with data and isolate
collection, Marc Izzo and Scott Gossweiler for their expert technical assistance, Richard Facklam and the CDC Streptococcal Laboratory for T typing results, and Theresa Hoenes for assistance with
DNA sequencing. We also thank Robin Ryder and Jim Hadler for
their support in these efforts.
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