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Journal of Clinical Microbiology, December 1999, p. 4194-4197, Vol. 37, No. 12
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Characterization of Blood Culture Isolates of
Streptococcus dysgalactiae subsp. equisimilis
Possessing Lancefield's Group A Antigen
Claudia M.
Brandt,1,*
Gerhard
Haase,1
Norbert
Schnitzler,1
Reinhard
Zbinden,2 and
Rudolf
Lütticken1
Institute of Medical Microbiology and
National Reference Center for Streptococci, University Hospital RWTH
Aachen, Aachen, Germany,1 and Department
of Medical Microbiology, University of Zürich, Zürich,
Switzerland2
Received 6 July 1999/Accepted 23 August 1999
 |
ABSTRACT |
For three human blood culture isolates of beta-hemolytic
streptococci with Lancefield's serogroup A antigen, phylogenetic analysis of the 16S rRNA genes confirmed biochemical identification as
Streptococcus dysgalactiae subsp. equisimilis.
Genes encoding M or M-like proteins, which are considered to be major
virulence determinants in streptococci, were detected in all of these
strains. Our data clearly demonstrate that for beta-hemolytic
streptococci, the species assignment should not be based on the results
of serogrouping alone.
| |
TEXT |
Streptococci were first serogrouped
by Rebecca C. Lancefield in 1933 on the basis of specific cell
wall-associated carbohydrates (16). The term group A
streptococcus has been used generally as a synonym for
Streptococcus pyogenes despite the fact that minute-colony-forming species of the "Streptococcus
milleri" group may also rarely possess Lancefield's serogroup A
antigen (12, 18). Moreover, Bert and Lambert-Zechovsky
(3) have recently published a case report of recurrent
bacteremia due to a biochemically identified strain of
Streptococcus dysgalactiae subsp. equisimilis exhibiting Lancefield's serogroup A antigen. This clinical isolate was
kindly provided by Bert and Lambert-Zechovsky for further investigation
and assigned streptococcal culture collection no. AC-2074. The present
report describes the phylogenetic analysis and molecular
characterization of blood culture isolates of S. dysgalactiae subsp. equisimilis belonging to
Lancefield's serogroup A.
(This work has been presented in part at the 9th European Congress of
Clinical Microbiology and Infectious Diseases, Berlin, Germany, 21 to
24 March 1999 [4a].)
Case 1.
A 78-year-old man presented to the hospital with acute
onset of headache, fever of 40°C, and chills. His medical history
included liver cirrhosis due to chronic hepatitis C, with signs of
portal hypertension. On physical examination, local erythema and
tenderness of his right foot were noted and the diagnosis of an
erysipelas was made. A minor skin lesion acquired 4 days prior to
admission probably served as the site of entry for bacteria. Blood
cultures were positive for beta-hemolytic streptococci, and
antimicrobial therapy with a standard dose of intravenous amoxicillin
was started. The patient's condition improved, and he was discharged 6 days later on an oral regimen to complete a 10-day course of
amoxicillin. One blood culture isolate was referred to the National
Reference Center for Streptococci, Aachen, Germany, and assigned
streptococcal culture collection no. AC-2713.
Case 2.
A 72-year-old woman who was undergoing diagnostic
workup for an indeterminate paraspinal mass (chronic inflammatory
lesion versus osteochondroma versus osteosarcoma) was referred for
symptoms consistent with acute tonsillitis. Blood was obtained, and
blood cultures grew beta-hemolytic streptococci. The patient was
treated according to the results for in vitro susceptibility testing
with cefuroxime. Twenty-four days later, the patient had fully
recovered. This clinical isolate was assigned streptococcal culture
collection no. AC-2832.
Methods.
Lancefield's group-specific streptococcal
carbohydrates were extracted from an overnight culture in
Todd- Hewitt broth according to the hot-formamide technique of
Fuller (8). Following extraction, serogroup was determined
by the Ouchterlony immunodiffusion technique with rabbit antisera
against the antigens A, C, G, and A-variant (14, 17).
Antigen-antibody precipitation lines were visualized by Coomassie blue
staining. In addition, trypsinized bacterial suspensions were tested
for agglutination with the lectin from Dolichos biflorus
(Biotrend, Cologne, Germany) in order to detect the group C
streptococcal antigen (15, 20, 23). The isolates were
biochemically identified with the Rapid ID 32 Strep API system (bioMérieux, Marcy-l'Etoile, France).
Streptococcal genomic DNA was prepared according to standard protocols
(2). The genomes of the strains were hybridized with an
oligonucleotide probe specific for S. pyogenes (Accuprobe; Gen-Probe Incorporated, San Diego, Calif.). Nearly the entire 16S rRNA
gene was amplified by PCR, and the resulting products were purified
with the Quiaquick PCR purification kit (Qiagen, Heiden, Germany) and
sequenced on an ABI 373 automated DNA sequencer (Applied Biosystems,
Weiterstadt, Germany) by following the protocol of Hiraishi
(10). The sequence editor DCSE, version 2.54 (5), was used for editing and concatenation and for multiple alignment of
the three strains' 16S ribosomal DNA (rDNA) sequences with those of
the following strains deposited in the EMBL database: S. pyogenes ATCC 12344 (accession no. AB002521); S. agalactiae ATCC 13813 (accession no. U02908); Streptococcus
dysgalactiae subsp. dysgalactiae ATCC 43078, serogroup
C (accession no. AB002485), and ATCC 27957, serogroup C (accession no.
AB002484); S. dysgalactiae subsp. equisimilis LMG
16026, serogroup C (accession no. AB008926); and S. pneumoniae MAFF 911410 (accession no. AB002522). The 16S rDNA
sequences of the three strains were also aligned with 16S rDNA
sequences obtained in our laboratory from the following strains of the
National Reference Center's collection: S. pyogenes AC-2892; S. agalactiae AC-2581; S. dysgalactiae
subsp. dysgalactiae GCS-1338, serogroup C; S. dysgalactiae subsp. equisimilis ATCC 35666, serogroup
C; and S. dysgalactiae subsp. equisimilis ATCC 12394, serogroup G. Phylogenetic studies were performed by using the
neighbor-joining method based on a dissimilarity matrix corrected for
multiple mutations per site, using the algorithm of Juke and Cantor.
Confidence values for individual branches were determined by bootstrap
analysis based on 1,000 bootstrap trees that were generated from
resampled characters. All calculations were performed with the software
package TREECON, version 1.3b (24).
The presence of genes encoding M- or M-like proteins was determined by
PCR with "all M" primers (19). The resulting products were purified and sequenced. The nucleotide sequence encoding the
N-terminal hypervariable portion of the M protein was subjected to
similarity searches against the National Institutes of Health DNA
database (1).
Remarks and conclusion.
It was shown unequivocally that all
three isolates exhibit the serogroup A antigen but not serogroup C, G,
or A-variant antigens. Results of the Ouchterlony immunodiffusion are
shown in Fig. 1. The
N-acetylgalactosamine specificity of the group C
carbohydrate antigen was not detected by agglutination with the lectin
from D. biflorus for any of the three strains tested.
Isolates AC-2713 and AC-2832 were biochemically identified as S. dysgalactiae subsp. equisimilis (API code 1512 2041 110; 99.5% identity; T = 1.0). As published previously for
isolate AC-2074 (3), our results revealed the following
characteristic properties to discriminate group A S. dysgalactiae subsp. equisimilis from S. pyogenes: production of 
-glucuronidase, fermentation of ribose,
and the absence of pyrrolidonylarylamidase production. A negative
Voges-Proskauer reaction provides a means to differentiate between the
large-colony-forming S. dysgalactiae subsp.
equisimilis and the minute-colony-forming species of the
S. milleri group. Hybridization with the Accuprobe oligonucleotide probe specific for S. pyogenes was not
detected for any of the three strains tested.
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FIG. 1.
Ouchterlony immunodiffusion of cell wall carbohydrate
extracts (outer wells) tested with rabbit antisera against the antigens
A, C, G, and A-variant (central wells). 1, serogroup-specific control
antigen; 2, AC-2074; 3, AC-2713; 4, AC-2832.
|
|
A rooted phylogenetic distance tree inferred by analysis of 16S rDNA
sequences is shown in Fig. 2. S. pneumoniae was arbitrarily chosen as the outgroup taxon. All three
blood culture isolates studied clustered with the type strain of
S. dysgalactiae subsp. equisimilis, LMG 16026, as
a sister group to S. dysgalactiae subsp. dysgalactiae in the phylogenetic tree. The phylogenetic
analyses revealed a substantial evolutionary distance between S. pyogenes and the S. dysgalactiae subsp.
equisimilis blood culture isolates exhibiting the Lancefield
serogroup A antigen.
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FIG. 2.
Rooted phylogenetic tree inferred by analysis of 16S
rDNA sequences of the three blood culture isolates AC-2074, AC-2713,
and AC-2832 and other members of the pyogenic streptococci by the
neighbor-joining method. Bootstrap values of  975 are displayed; nodes
with a support of <975 are drawn unresolved.
|
|
For the three strains studied, genes (emm or
emmL) encoding M- or M-like proteins were detected. The
respective nucleotide sequences of strains AC-2713 and AC-2832 revealed
a high degree of similarity (>80% identity for a portion of at least
200 bp within the 5' region of the respective gene) to the genotype b emmL gene described previously for serogroup G S. dysgalactiae subsp. equisimilis (21); the
emm or emmL gene sequence of strain AC-2074
showed a high degree of similarity to the emm41 gene of S. pyogenes.
Pyogenic streptococci have been divided conventionally on the basis of
their Lancefield group antigen, habitat, pathogenicity, and biochemical
characteristics (7, 16). With the availability of automated
DNA sequencing and a rapidly growing database, the analysis of 16S rRNA
sequences has become one of the most powerful tools for phylogenetic
analysis and species identification. The finding that the 16S rDNA
sequences of all three strains studied are closely related to that of
the type strain of S. dysgalactiae subsp.
equisimilis suggests that biochemical methods, like the API
system, are an appropriate diagnostic approach for the identification of this species. The observation of a substantial evolutionary distance
between S. dysgalactiae subsp. equisimilis and
S. pyogenes is in agreement with results of previous
phylogenetic analyses of beta-hemolytic streptococci (13)
and supports the application of an oligonucleotide probe specific for
S. pyogenes in the identification of beta-hemolytic
streptococci exhibiting Lancefield's serogroup A antigen. These data
clearly show that for species assignment of beta-hemolytic
streptococci, the determination of the serogroup is insufficient as the
sole diagnostic tool, as outlined previously for group G streptococci
(9).
In humans, S. dysgalactiae subsp. equisimilis may
colonize the skin, throat, and genital tract. Recently, this species
has been isolated with an increasing frequency as the cause of various human infections, such as cellulitis, pharyngitis, sepsis, meningitis, and endocarditis (6). Just as most patients with infections due to S. dysgalactiae subsp. equisimilis
exhibiting serogroup C or G antigen have underlying diseases,
predominantly malignancies (4, 22), the three patients
diagnosed with infections due to group A S. dysgalactiae
subsp. equisimilis also suffered from compromising
underlying diseases. The finding of two additional patients with
bacteremia due to S. dysgalactiae subsp.
equisimilis belonging to Lancefield's serogroup A and the
detection of emm or emmL genes encoding a major
virulence factor in all three strains tested provide evidence that
strains of S. dysgalactiae subsp. equisimilis of
Lancefield's serogroup A are probably as pathogenic as those
exhibiting Lancefield's serogroup C or G antigen. Therefore, it can be
concluded that beta-hemolytic streptococci that possess the serogroup A
antigen but are negative for the Voges-Proskauer reaction and do not
produce pyrrolidonylarylamidase can be found in serious human
infections and may be identified as S. dysgalactiae subsp.
equisimilis.
| |
ACKNOWLEDGMENTS |
We thank F. Bert (Clichy, France), M. Jacobs (Dillingen, Germany),
and C. Jochum (Püttlingen, Germany) for their cooperation and the
referral of the strains to the National Reference Laboratory for
Streptococci. We appreciate the excellent technical assistance of M. Breuer-Werle and B. Weidenhaupt.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Medical Microbiology, National Reference Center for Streptococci,
University Hospital RWTH Aachen, Pauwelsstraße 30, D-52074 Aachen,
Germany. Phone: 49-241-8089512. Fax: 49-241-8888483. E-mail:
rluetticken{at}post.klinikum.rwth-aachen.de.
| |
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Journal of Clinical Microbiology, December 1999, p. 4194-4197, Vol. 37, No. 12
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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