Taxonomic dissection of the Streptococcus bovis group by analysis of manganese-dependent superoxide dismutase gene (sodA) sequences: reclassification of 'Streptococcus infantarius subsp coli' as Streptococcus lutetiensis sp nov and of Streptococcus bovis biotype II.2 as Streptococcus pasteurianus sp nov.

Abstract

The taxonomic dissection of the Streptococcus bovis-Streptococcus equinus group was carried out upon obtaining sequences for the manganese-dependent superoxide dismutase gene (sodA) of the type strains of S. bovis, Streptococcus caprinus, S. equinus, Streptococcus gallolyticus, Streptococcus infantarius, Streptococcus macedonicus and Streptococcus waius. The sodA sequences of 29 streptococcal strains of animal and human origin that were related to S. bovis were also sequenced. A phylogenetic analysis of the sodA sequences revealed that the S. bovis-S. equinus group comprises five different clusters that correspond to five distinct species. The type strains of S. bovis and S. equinus were associated in the same cluster, corresponding to the species S. equinus. The type strains of S. caprinus, S. gallolyticus, S. macedonicus and S. waius were associated in the same cluster, which defined a single species containing S. gallolyticus and its junior synonym S. caprinus, and S. macedonicus and its junior synonym S. waius. The two subspecies thought to constitute the species S. infantarius, namely S. infantarius subsp. infantarius and 'S. infantarius subsp. coli', were located in two distinct clusters. One of these clusters defined the species S. infantarius and included the type strain of S. infantarius subsp. infantarius. The other cluster defined 'S. infantarius subsp. coli', leading to the proposal of its reclassification as the novel species Streptococcus lutetiensis (NEM 782T = CIP 106849T). The remaining cluster comprised all of the strains previously identified as belonging to S. bovis biotype 11.2, leading to the proposal to reassign these strains to the novel species Streptococcus pasteurianus (NEM 1202T = CIP 107122T). The results of the phylogenetic analysis were confirmed by DNA-DNA hybridization experiments, thus demonstrating that sequence databases of defined DNA targets, such as sodA, may constitute a valuable alternative approach for modern bacterial systematics.

Full text

International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1247–1255 DOI: 10.1099/ijs.0.02044-0
Taxonomic dissection of the Streptococcus
bovis group by analysis of manganese-
dependent superoxide dismutase gene (sodA)
sequences: reclassification of ‘Streptococcus
infantarius subsp. coli’asStreptococcus
lutetiensis sp. nov. and of Streptococcus bovis
biotype II.2 as Streptococcus pasteurianus sp.
nov.
Laboratoire Mixte Pasteur-
Necker de Recherche sur les
Streptocoques et
Streptococcies and Unite
!
INSERM 411, Faculte
!de
Me
!decine Necker-Enfants
Malades, 75730 Paris Cedex
15, France
Claire Poyart, Gilles Quesne and Patrick Trieu-Cuot
Author for correspondence: Claire Poyart. Tel: j33 (1) 40 61 56 79. Fax : j33 (1) 40 61 55 92.
e-mail: cpoyart!pasteur.fr
The taxonomic dissection of the Streptococcus bovis–Streptococcus equinus
group was carried out upon obtaining sequences for the manganese-dependent
superoxide dismutase gene (sodA) of the type strains of S. bovis,Streptococcus
caprinus,S. equinus,Streptococcus gallolyticus,Streptococcus infantarius,
Streptococcus macedonicus and Streptococcus waius. The sodA sequences of 29
streptococcal strains of animal and human origin that were related to S. bovis
were also sequenced. A phylogenetic analysis of the sodA sequences revealed
that the S. bovis–S. equinus group comprises five different clusters that
correspond to five distinct species. The type strains of S. bovis and S. equinus
were associated in the same cluster, corresponding to the species S. equinus.
The type strains of S. caprinus,S. gallolyticus,S. macedonicus and S. waius were
associated in the same cluster, which defined a single species containing S.
gallolyticus and its junior synonym S. caprinus, and S. macedonicus and its
junior synonym S. waius. The two subspecies thought to constitute the species
S. infantarius, namely S. infantarius subsp. infantarius and ‘S. infantarius subsp.
coli’, were located in two distinct clusters. One of these clusters defined the
species S. infantarius and included the type strain of S. infantarius subsp.
infantarius. The other cluster defined ‘S. infantarius subsp. coli’, leading to the
proposal of its reclassification as the novel species Streptococcus lutetiensis
(NEM 782TlCIP 106849T). The remaining cluster comprised all of the strains
previously identified as belonging to S. bovis biotype II.2, leading to the
proposal to reassign these strains to the novel species Streptococcus
pasteurianus (NEM 1202TlCIP 107122T). The results of the phylogenetic analysis
were confirmed by DNA–DNA hybridization experiments, thus demonstrating
that sequence databases of defined DNA targets, such as sodA, may constitute a
valuable alternative approach for modern bacterial systematics.
Keywords: Streptococcus bovis,Streptococus lutetiensis sp. nov., Streptococcus
pasteurianus sp. nov., superoxide dismutase gene (sodA), 16S rRNA gene
.................................................................................................................................................................................................................................................................................................................
Published online ahead of print on 29 November 2001 as DOI 10.1099/ijs.0.02044-0.
Abbreviation: sodAint, internal fragment of sodA.
The GenBank accession numbers for the sodAint sequences reported in this study can be found in Table 1. The GenBank accession numbers for the 16S rDNA
sequences of S. lutetiensis NEM 782Tand S. pasteurianus NEM 1202Tare AJ297189 and AJ297195, respectively.
02044 #2002 IUMS Printed in Great Britain 1247

C. Poyart, G. Quesne and P. Trieu-Cuot
INTRODUCTION
Streptococcus bovis is a normal inhabitant of the
ruminant and human gut. In humans, it has been
reported to be the causative agent of meningitis,
septicaemia and endocarditis, and numerous reports
have suggested a potential relationship between in-
creased faecal carrier levels of S.bovis and human
gastrointestinal disease (Duval et al., 2001; Grant et
al., 2000; Manfredi et al., 1999 ; Zarkin et al., 1990).
Therefore, the correct identification of S.bovis isolates
is important in clinical microbiology laboratories.
The taxonomic status of S.bovis strains has been
evolving in the last few decades and has progressively
changed according to the description of new species
originally identified as S.bovis. In the 1990s, four new
species were described, Streptococcus gallolyticus
(Osawa et al., 1995), Streptococcus macedonicus (Tsak-
alidou et al., 1998), Streptococcus waius (Flint et al.,
1999) and Streptococcus infantarius (Bouvet et al.,
1997). In clinical laboratories, the accurate identifica-
tion of these streptococci is based on phenotypic
characteristics that permit the classification of S.bovis
strains into two biotypes (Facklam et al., 1984 ; Knight
& Schlaes, 1985; Ruoff et al., 1984, 1989). However,
these phenotypic characterizations are impaired due to
the variable expression of certain traits and because of
the frequent ambiguity in the interpretation of such
data. Consequently, nucleic-acid-based technologies,
such as DNA–DNA hybridization or the amplification
of selected targets, have been developed to complement
and improve the identification of streptococci at the
species level (Garnier et al., 1997 ; Kawamura et al.,
1995, 1999; Poyart et al., 1998). Farrow et al. (1984)
demonstrated that on the basis of DNA–DNA hybridi-
zation data S.bovis strains could be classified into six
genomic groups that exhibited between 40 and 60%
DNA similarity with each other. These authors also
demonstrated that biotype I strains were genotypically
homogeneous and distinct from biotype II strains,
which include the type strains of S.bovis and Strep-
tococcus equinus. More recently, based on 16S rDNA
sequence analysis, Clarridge et al. (2001) have sug-
gested that S.bovis biotype II.2 strains constitute a
separate genospecies that is distinct from S.bovis,S.
gallolyticus and S.infantarius.
The interpretation of 16S rDNA sequence data may be
complicated by the fact that divergent 16S rDNA
sequences may exist within a single organism (Ueda et
al., 1999) or, alternatively, by the fact that closely
related species may have nearly identical 16S rDNA
sequences (Fox et al., 1992). The latter has been shown
for members of the genus Streptococcus, namely
Streptococcus pneumoniae,Streptococcus mitis and
Streptococcus oralis (Kawamura et al., 1995). The 16S
rDNA sequences of the type strains of the S.bovis
group (S.bovis,Streptococcus caprinus,S.equinus,S.
gallolyticus,S.infantarius and S.macedonicus) exhibit
a percentage of identity ranging from 97n1 (e.g. S.
equinus and S.gallolyticus, and S.gallolyticus and S.
infantarius)to99n8% (S.bovis and S.infantarius). To
differentiate such strains, it is possible to use alternative
single-copy target sequences that exhibit greater se-
quence divergence than that of 16S rDNA. The sodA
gene of the Gram-positive cocci, which encodes the
manganese-dependent superoxide dismutase (Mn-
SOD), fulfils these criteria. We have previously de-
scribed a PCR assay, based on the utilization of
degenerate primers, which enabled the amplification of
an internal fragment representing approximately 83 %
of the sodA gene encoding Mn-SOD in various Gram-
positive bacteria, including streptococci and entero-
cocci (Poyart et al., 1995). We have also reported that
sequencing the sodA PCR product, with the same
degenerate primers, constitutes a valuable approach to
the genotypic identification of species belonging to the
genera Streptococcus and Enterococcus (Poyart et al.,
1995, 1998, 2000). This target gene has also been used
for the identification of other bacteria at the species
level, including coagulase-negative staphylococci
(Poyart et al., 2001) and mycobacteria (Zolg &
Philippi-Schulz, 1994). In this work, we carried out a
taxonomic analysis of the S.bovis group, by using the
same approach as described previously and demon-
strated the usefulness of a sodA-based database for the
species identification of strains belonging to the S.
bovis–S.equinus complex. Furthermore, phylogenetic
studies of sodA gene sequences and DNA–DNA
hybridization experiments support the recognition of
two distinct novel species within the genus Strep-
tococcus, for which the names Streptococcus lutetiensis
(formerly ‘S.infantarius subsp. coli’) and Streptococcus
pasteurianus (formerly S.bovis biotype II.2) are pro-
posed.
METHODS
Bacterial strains and culture conditions. The main charac-
teristics of the strains used in this study, including the type
strains, are listed in Table 1. The isolates of S.bovis were of
various origins and were collected over a period of at least 10
years. All strains were grown at 37 mC on Columbia horse
blood agar (bioMe
!rieux) or in brain-heart infusion (BHI)
broth under anaerobic conditions. Cultures were stored at
k80 mC in BHI broth (Difco) supplemented with 10 % (w\v)
glycerol until required.
Phenotypic characteristics. The strains were characterized
for their morphological, growth and biochemical properties.
The production of acetoin, enzymic reactions and fermen-
tation of carbohydrates were determined using the API
20Strep and Rapid ID 32Strep systems, according to the
manufacturer’s recommendations (bioMe
!rieux). All strains
were tested for growth on agar plates supplemented with
40% bile\aesculin, 5 % sucrose or 0n04 % sodium tellurite.
Growth was tested in broth containing 6n5% (w\v) NaCl
and gas production was assayed in MRS broth (Bio-Rad).
The presence of the Lancefield’s group D antigen was
determined with the Streptex test, according to the manu-
facturer’s recommendations (bioMe
!rieux).
PFGE. High-molecular-mass DNA from the growth obtained
from a single plate for each strain was extracted in agarose
plugs by conventional methods, digested with SmaI and
1248 International Journal of Systematic and Evolutionary Microbiology 52

Dissection of S.bovis group using sodA sequences
Table 1. Characteristics of the streptococcal strains used in this study
Species* Source Rapid ID
32Strep
biotype
sodAint
cluster†
sodAint-based
identification
GenBank no. for
sodAint sequence
Type strains
S.alactolyticus CIP 103244TPig intestine  –S.alactolyticus Z95894
S.bovis CIP 102302TCow faeces  CS.bovis Z95896
S.caprinus CIP 104887TGoat rumen  ES.caprinus AJ297182
S.equinus CIP 102504THorse faeces  CS.equinus Z95903
S.gallolyticus CIP 105428TKoala faeces  ES.gallolyticus AJ297183
S.infantarius subsp. infantarius CIP 103233TInfant faeces  AS.infantarius subsp. infantarius AJ297184
S.macedonicus CIP 105683TKasseri cheese  ES.macedonicus AJ297186
S.salivarius CIP 102503THuman blood  –S.salivarius Z95916
S.waius CIP 106079TMilk  ES.waius AJ297187
Clinical isolates
S.bovis NEM 760 Infant faeces 22053003110 B S.lutetiensis AJ297188
S.bovis NEM 782T, CIP 106849T‡Human isolate 22053003110 B S.lutetiensis AJ297189
S.bovis NEM 1101, CIP 103567 Human isolate 22273063150 E S.gallolyticus AJ297190
S.bovis NEM 1195, CIP 105064 Human isolate 22273063150 E S.gallolyticus AJ297191
S.bovis NEM 1196, CIP 105065 Human blood 22273063150 E S.gallolyticus AJ297192
S.bovis NEM 1197, CIP 105066 Human blood 22273063150 E S.gallolyticus AJ297193
S.bovis NEM 1201, CIP 105069 Human blood 22273063150 E S.gallolyticus AJ297194
S.bovis NEM 1202T, CIP 107122T§Human cerebrospinal fluid 63077003150 D S.pasteurianus AJ297195
S.bovis NEM 1203, CIP 105071 Human blood 22273063150 E S.gallolyticus AJ297196
S.bovis NEM 1204, CIP 105072 Human blood 22273063150 E S.gallolyticus AJ297197
S.bovis NEM 1205, CIP 105073 Human blood 63077203150 D S.pasteurianus AJ297198
S.bovis NEM 1206, CIP 105074 Human blood 63077201150 D S.pasteurianus AJ297199
S.bovis NEM 1227, CIP 105068 Human blood 63077003150 D S.pasteurianus AJ297200
S.bovis NEM 1350, CIP 105284 Foal faeces 22273063150 E S.gallolyticus AJ297201
S.bovis NEM 1351, CIP 105285 Foal faeces 22273063150 E S.gallolyticus AJ297202
S.bovis NEM 1352, CIP 105286 Foal faeces 22273063150 E S.gallolyticus AJ297203
S.bovis NEM 1353, CIP 105287 Dog uterus 22273063150 E S.gallolyticus AJ297204
S.bovis NEM 1603 Human cerebrospinal fluid 22053003110 B S.lutetiensis AJ297205
S.bovis NEM 1771 Human urine 63077001150 D S.pasteurianus AJ297206
S.bovis NEM 1773 Human blood 22273063150 E S.gallolyticus AJ297207
S.bovis NEM 1772 Human blood 22273063150 E S.gallolyticus AJ297208
S.bovis NEM 1774, CIP 105070 Human cerebrospinal fluid 63077003150 D S.pasteurianus AJ297209
S.bovis NEM 1775, CIP 103560 Human isolate 22273063150 E S.gallolyticus AJ297210
S.equinus NEM 1761, CIP 103232 Milk, bovine mastitis 22273063150 E S.gallolyticus AJ297211
S.equinus NEM 1764, CIP 56.23 Human isolate 02013001100 B S.lutetiensis AJ297212
S.equinus NEM 1760, CIP 82.5 Horse faeces 20003001110 C S.equinus AJ297213
‘S.infantarius subsp. coli ’ NEM 1867, NCDO 964 Unknown source 22053001110 B S.lutetiensis AJ306978
S.infantarius subsp. infantarius NEM 1868, CIP 106106 Infant faeces 2205306610 A S.infantarius AJ306979
S.infantarius subsp. infantarius NEM 1869, CIP 106107 Human blood 0205306610 A S.infantarius AJ306980
, Not determined.
* CIP, Collection de l ’Institut Pasteur; NEM, Necker-Enfants Malades ; NCDO, National Collection of Dairy Organisms.
†The sodAint cluster refers to the divisions shown in Fig. 1.
‡Type strain of S.lutetiensis.
§Type strain of S.pasteurianus.
BssHII and separated through a 1 % agarose gel by using
a clamped-homogeneous-field electrophoresis apparatus
(CHEF MAPPER DRII, Bio-Rad), as described previously
(Poyart et al., 1997).
DNA–DNA hybridization. Genomic DNA was extracted as
described previously (Poyart et al., 1997). DNA samples
were diluted in twofold serial dilutions to provide concen-
trations of between 0n0625 and 1 µg DNA (100 µl)−". Six
replicate 100 µl samples of each dilution were loaded onto
Nylon blotting membranes (Hybond-N+; Amersham) using
a dot-blotting apparatus. Probes were prepared for six
Streptococcus strains (S.bovis CIP 102302T,S.gallolyticus
CIP 105428T,S.macedonicus CIP 105683T,S.infantarius
subsp. infantarius CIP 103233T,S.lutetiensis NEM 782Tand
S.pasteurianus NEM 1202T) by labelling them with $#P using
the Megaprime DNA Labelling System (Amersham). Hybri-
dizations were performed as follows. Prehybridization and
hybridization were carried out for 2 h and 18 h, respectively,
at 65 mC in 10 ml of Rapid-hyb buffer (Amersham), followed
by two washes in 2iSSC (1n5 M NaCl, 0n15 M trisodium
citrate) containing 0n1 % SDS at 65 mC for 15 min and by two
washes in 1iSSC containing 0n1 % SDS at 65 mC for 15 min.
Images were revealed with a STORM phosphoimager
(Molecular Dynamics) and interpreted with the software
XdotsReader (COSE), which quantified the intensity of the
signal associated with each dot.
PCR amplification and sequencing. The rapid extraction of
bacterial genomic DNA collected from 2 ml of an overnight
culture was performed with the InstaGene Matrix (Bio-
Rad). The sodA degenerate primers d1 (5h-CCITAYICITA-
YGAYGCIYTIGARCC-3h) and d2 (5h-ARRTARTAIGC-
RTGYTCCCAIACRTC-3h) were used to amplify an in-
ternal fragment of sodA (sodAint), representing approxi-
mately 82% of the complete sodA gene. These primers
match at positions 25–51 (d1) and 487–510 (d2) of the 609 bp
long sodA gene of the S.bovis type strain, which was taken
as a reference. PCRs were performed on a Gene Amp System
2400 thermal cycler (Perkin Elmer) in a final volume of 50 µl
http://ijs.sgmjournals.org 1249

C. Poyart, G. Quesne and P. Trieu-Cuot
containing 250 ng of DNA as template, 0n5µM of each
primer, 200 µM of each dNTP and 1 U of AmpliTaq Gold
DNA polymerase (Perkin Elmer) in a 1iamplification
buffer [10 mM Tris\HCl (pH 8n3), 50 mM KCl, 1n5mM
MgCl#]. The PCR mixtures were denatured (3 min at 95 mC)
and then subjected to 30 cycles of amplification (60 s of
annealing at 37 mC, 60 s of elongation at 72 mC and 30 s of
denaturation at 95 mC). PCR products were purified on a S-
400 Sephadex column (Pharmacia) and directly sequenced
on both strands with the degenerate primers d1 and d2 by
using the ABI-PRISM BigDye Terminator Sequencing Kit
and a Genetic ABI-PRISM 310 Sequencer Analyser (Perkin
Elmer), as described previously (Poyart et al., 2000). De-
termination of the 16S rDNA sequences was done as follows.
The PCR fragments obtained by using the pairs of primers
R1 and R2 (R1, R2, R7 and R8), R3 and R4 (R3, R4, R9 and
R10) and R5 and R6 (R5, R6, R11 and R12) were sequenced
on both DNA strands by using the primers indicated in
parentheses, as described previously (Poyart et al., 2000).
The resulting sequences were assembled to generate a single
contig of approximately 1450 bp that corresponded to the
16S rDNA sequence. The sequences of the primers used
were: R1, 5h-TAACACATGCAAGTCGAACG-3h; R2, 5h-
CCTGCGCTCGCTTTACGCCC-3h; R3, 5h-GTGCCAG-
CAGCCGCGGTAAT-3h; R4, 5h-ACACGAGCTGACG-
ACAGCCA-3h; R5, 5h-GGGGGCCCGCACAAGCGG-3h;
R6, 5h-AGGAGGTGATCCAACCGCA-3h; R7, 5h-GGCC-
ACGATGCATAGCCG-3h; R8, 5h-GACTGCTGCCTCC-
CGTAG-3h; R9, 5h-CTGAGGCTCGAAAGCGTGGG-3h;
R10, 5h-CCCACGCTTTCGAGCCTCAG-3h; R11, 5h-GA-
GGAAGGTGGGGATGACGT-3h; R12, 5h-CGTCATCC-
CCACCTTCCTCC-3h.
Sequence analysis. The nucleotide sequences were analysed
with Perkin Elmer software (Sequence Analysis, Sequence
Navigator and AutoAssembler). Multiple alignments of the
sodA and 16S rRNA gene sequences were carried out with
the   program (Jeanmougin et al., 1998). The
construction of the unrooted phylogenetic trees was per-
formed with both the neighbour-joining (Saitou & Nei,
1987) and the maximum-parsimony (Fitch, 1971) methods,
using the  package (version 3.57c ; Felsenstein, 1995).
The reliability of the tree nodes was evaluated by calculating
the percentage of 1000 bootstrap re-samplings that sup-
ported each topological element. Phylogenetic trees were
also generated based on the translated partial SodA protein
sequences.
RESULTS AND DISCUSSION
By using the primers d1 and d2, we amplified and
sequenced sodAint from the type strains of S.bovis,S.
caprinus,S.equinus,S.gallolyticus,S.infantarius,S.
macedonicus and S.waius (Table 1). In this study, we
also included 29 streptococcal strains of animal and
human origin that were originally identified as S.
bovis–S.equinus by conventional microbiological tests.
The strains used in this study were analysed by PFGE
after digestion with SmaI and BssHII. The analysis of
the restriction profile patterns obtained with these two
enzymes for all the strains studied were different from
each other by at least five fragments, demonstrating
their unrelatedness (data not shown).
A single DNA fragment, corresponding to the expect-
ed 480 bp amplification product sodAint, was observed
in all cases following agarose-gel electrophoresis and
ethidium-bromide staining (data not shown). Analysis
of the sequences of these amplicons revealed that they
were actually fragments of sodA, since the corre-
sponding deduced polypeptides revealed that they all
possessed three histidyl residues and one aspartyl
residue, supposedly serving as metal ligands at posi-
tions characteristic of manganese- or iron-dependent
superoxide dismutases (Parker & Blake, 1988a, b). A
multiple alignment of the streptococcal sodAint se-
quences was carried out by using the  
program. The sequences of the degenerate primers d1
and d2 and alignment gaps were not taken into
consideration for calculations. Phylogenetic analyses
of the sodAint sequences (430 bp) were performed
using both the neighbour-joining and maximum-
parsimony methods, as contained within the 
software package (version 3.57c; Felsenstein, 1995).
The consensus trees derived from the two methods
were virtually identical, hence only the consensus tree
constructed by the neighbour-joining method is shown
here (Fig. 1). This phylogenetic tree revealed that the
S.bovis group can be divided into five major clusters
(A, B, C, D and E; Fig. 1), which were supported by
significant bootstrap values. The sodAint sequence
identity within each cluster was greater than 97n7%
(Fig. 1), whereas it varied from 80 to 96% if one
considered a pair of sequences from two strains
belonging to different clusters (data not shown). The
consensus tree based on SodA protein sequences had a
similar topology but the reliability of the tree nodes,
determined by a bootstrap analysis, was weaker than
that in the DNA consensus trees (data not shown).
These findings reflect the fact that DNA sequences are
generally more divergent than those of the corre-
sponding protein sequences, due to the degeneracy of
the genetic code.
Cluster A (Fig. 1) comprises three strains formerly
identified as S.infantarius subsp. infantarius, including
the type strain of the species S.infantarius (Bouvet et
al., 1997; Schlegel et al., 2000). The sodAint sequences
of these three strains displayed less than 1% sequence
divergence. The species S.infantarius, which belongs
to biotype II.1, had been previously identified on the
basis of DNA–DNA hybridization and ribotyping and
was shown to contain two subgroups, leading to the
description of two subspecies, S.infantarius subsp.
infantarius and ‘S.infantarius subsp. coli’ (Schlegel et
al., 2000).
Cluster B (Fig. 1) includes the strain previously defined
as ‘S.infantarius subsp. coli’ (Schlegel et al., 2000).
Comparison of the sodAint sequence of ‘S.infantarius
subsp. coli’ NEM 1867 with those of the S.infantarius
subsp. infantarius strains (Cluster A) revealed 10n3%
sequence divergence between the two clusters. These
results are in agreement with those obtained from
DNA–DNA hybridization experiments which demon-
strated that the two subspecies displayed less than
70% homology (Table 2) ; 70 % homology is in-
dicative of two strains belonging to the same species
1250 International Journal of Systematic and Evolutionary Microbiology 52

Dissection of S.bovis group using sodA sequences
S. salivarius CIP 102503T
S. alactolyticus CIP 103244T
S. infantarius subsp. infantarius CIP 103233T
NEM 1868
NEM 1869
NEM 1603
NEM 760
NEM 1764
100
‘S. infantarius subsp. coli’ NEM 1867
100
99
87
S. lutetiensis NEM 782T
S. bovis CIP 102302T
NEM 1760
S. equinus CIP 102504T
NEM 1227
NEM 1774
S. pasteurianae NEM 1202T
NEM 1205
NEM 1771
NEM 1206
S. caprinus CIP 104887T
NEM 1352
S. macedonicus
S. waius CIP 106079T
NEM 1775
NEM 1201
NEM 1773
NEM 1350
NEM 1353
NEM 1203
NEM 1204
S. gallolyticus CIP 105428T
NEM 1197
NEM 1101
NEM 1195
NEM 1196
NEM 1761
NEM 1772
NEM 1351
10 %
Cluster E
≥97·7
Cluster D
≥98·9
Cluster C
≥97·9
Cluster B
≥99·8
Cluster A
≥99·9
CIP 105683T
II.1
II.2
I
100
100
91
100
Biotype
sodAint
identity
(%)
.....................................................................................................
Fig. 1. Phylogenetic tree showing the
relationships among the sodAint sequences
from various streptococcal strains. The tree
was constructed using the neighbour-joining
method, and the sodAint sequences of the
type strains of S. salivarius and S. alactoly-
ticus were used as an outgroup to root the
tree. Relevant bootstrap values, expressed
as a percentage of 1000 replications, are
indicated at the appropriate nodes. The
scale bar (neighbour-joining distance) repre-
sents the percentage sequence divergence.
The accession numbers for the sodAint
sequences used in this study can be found
in Table 1.
Table 2. DNA–DNA hybridization values among species belonging to the S. bovis–S. equinus complex
.................................................................................................................................................................................................................................................................................................................
Species: 1, S.infantarius subsp. infantarius CIP 103233T;2,S.lutetiensis NEM 782T;3,S.bovis CIP 102302T;4,S.pasteurianus
NEM 1202T;5,S.gallolyticus CIP 105428T;6,S.macedonicus CIP 105683T. The sodAint cluster refers to the divisions shown in
Fig. 1.
Species sodAint
cluster
Homology when following DNAs were used as probes (%)
123456
S.infantarius CIP 103233TA 100 61n67 49n27 46n41 34n92 44n08
S.lutetiensis NEM 782TB62n78 100 50n07 53n24 40n57 42n9
S.bovis CIP 102302TC46n649n97 100 40n21 38n33 32n78
S.equinus CIP 102504TC51n14 48n77 91n03 49n16 39n61 40n65
S.pasteurianus NEM 1202TD43n34 37n83 37n76 100 60n38 56n63
S.gallolyticus CIP 105428TE35n79 38n58 42n97 60n67 100 69n09
S.caprinus CIP 104887TE34n59 38n143n83 57n12 97n570n31
S.macedonicus CIP 105683TE43n09 39n95 42n53 56n85 66n3 100
S.waius CIP 106079TE54n26 41n75 40n05 55n12 68n45 93n33
(Wayne et al., 1987). Taken together, these data
demonstrate that S.infantarius subsp. infantarius and
‘S.infantarius subsp. coli’ do not belong to the same
species. Cluster B also contains four human clinical
strains identified as belonging to S.bovis biotype II.1
and one strain (NEM 1764) initially identified by
phenotypic tests as S.equinus. All of the strains
belonging to this cluster possessed a similar Rapid ID
32Strep biotype and exhibited less than 0n2 % di-
vergence between their sodAint sequences (Table 1 and
data not shown). Thus, we propose the description of
the strains belonging to this cluster as members of the
http://ijs.sgmjournals.org 1251

C. Poyart, G. Quesne and P. Trieu-Cuot
novel species Streptococcus lutetiensis, which will
incorporate ‘S.infantarius subsp. coli’.
Cluster C (Fig. 1) is composed of three strains,
including the type strains of S.bovis and S.equinus.
The sodAint sequences of these strains displayed less
than 1% divergence. DNA–DNA hybridization ex-
periments confirmed that strains belonging to this
cluster were highly homologous to each other (90%)
but that they were poorly related to strains belonging
to the other clusters (53 % ; Table 2). These data
confirm previous results (Farrow et al., 1984) which
indicated that these bacteria represent a single species.
According to Rule 24b(2) of the Bacteriological Code
(1990 revision) (Lapage et al., 1992), we propose to
designate all strains belonging to Cluster C as S.
equinus. Among the 29 clinical isolates characterized in
this study, only one strain (NEM 1760) was assigned to
this cluster. This strain, isolated from horse faeces, was
identified as S.equinus on the basis of its Rapid ID
32Strep biotype. These results confirm previous asser-
tions that isolates of the species S.equinus, including
its heterotypic synonym S.bovis, are rarely encoun-
tered among human clinical isolates (Devriese et al.,
1998; Farrow et al., 1984 ; Nelms et al., 1995 ; White-
head & Cotta, 2000).
Cluster D (Fig. 1) encompasses six human clinical
isolates identified by phenotypic methods as belonging
to S.bovis biotype II.2 and it does not include any of
the type strains used in this study. The sodAint
sequences of these strains were highly similar (1n1%
divergence). In addition, their Rapid ID 32Strep
biotypes were nearly identical and differed from those
of strains belonging to the other clusters (Table 1).
DNA–DNA hybridization experiments performed
with NEM 1202Tused as a probe yielded low hybridi-
zation levels (38–61%) with the other type strains
studied (Table 2). These results are in agreement with
those obtained previously from DNA–DNA hybridi-
zation assays, ribotyping and analyses of 16S rRNA
gene sequences (Clarridge et al., 2001; Farrow et al.,
1984; Schlegel et al., 2000) and support the description
of a novel species within the S.bovis–S.equinus
complex. The name S.pasteurianus is proposed for this
novel species. A remarkable phenotypic characteristic
of the members of this new species is that they all
produced activity for all of the enzymes involved in
sugar metabolism (i.e. β-glucosidase, α-galactosidase,
β-glucuronidase, β-galactosidase and β-mannosidase)
tested in the API 20Strep and Rapid ID 32Strep
systems (Tables 1 and 3). It is also worth noting that
among the six unrelated clinical isolates which define
this cluster two were isolated from cerebrospinal fluid
and were responsible for meningitis. This observation
is consistent with a recent report in which all strepto-
coccal strains belonging to Lancefield’s group D
responsible for central nervous system infections were
identified as S.bovis biotype II.2 and clustered in the
same group (Clarridge et al., 2001). Further investiga-
tions are required to determine whether the strains
belonging to this species possess specific virulence
genes responsible for this neuropathogenicity.
Cluster E (Fig. 1) contains the type strains of S.
gallolyticus,S.caprinus,S.macedonicus and S.waius
(Brooker et al., 1994; Flint et al., 1999 ; Osawa et al.,
1995; Tsakalidou et al., 1998), and 15 strains dis-
playing the same Rapid ID 32Strep biotype and
assigned as S.bovis biotype I. The sodAint sequences of
these strains are highly similar and show less than
2n3% divergence. The fact that the sodAint sequences
of the S.gallolyticus and S.caprinus type strains are
almost identical (98n9%) confirms that these species
are subjective synonyms, as reported by Sly et al.
(1997). It is worth noting that the strains of human (n
l10) and animal (nl5) origin could not be differ-
entiated by this genotypic method (Table 1 and Fig. 1).
Conversely, the complete sequence identity observed
between the sodAint sequences of the type strains of S.
macedonicus and S.waius suggests that they should be
associated in a single species (Fig. 1). The validity of
these sequencing data are confirmed by the high
hybridization levels observed between S.macedonicus
and S.waius (93n3% ; Table 2). Accordingly, besides
the fact that they were both isolated from dairy
products, these two streptococcal strains share many
common phenotypic characters and their 16S rRNA
genes possess less than 0n2% sequence divergence, as
described by Flint et al. (1999), Tsakalidou et al. (1998)
and this work. Therefore, according to Rule 24b(2) of
the Bacteriological Code (1990 revision) (Lapage et al.,
1992), S.macedonicus should have nomenclatural
priority. It is therefore possible that Cluster E contains
two distinct species, S.gallolyticus and S.macedonicus.
Alternatively, it may contain a single species, S.
gallolyticus, and in this case S.macedonicus may
constitute an aesculin-negative variant of this species.
Our DNA–DNA hybridization data, which revealed
that the genomes of S.macedonicus\S.waius and S.
gallolyticus\S.caprinus display 70% homology,
favours this latter hypothesis (Table 2).
The sequences of the 16S rRNA genes of S.bovis,
S.caprinus,S.equinus,S.gallolyticus,S.infantarius
subsp. infantarius and S.macedonicus have been
published previously. We therefore determined the 16S
rDNA sequences of three strains (NEM 760, NEM
782Tand NEM 1603) and two strains (NEM 1202T
and NEM 1205) belonging to Clusters B and D,
respectively. Sequence analysis revealed that within
both of these clusters the 16S rDNA sequences were
identical (data not shown). The sequences of the 16S
rDNA of the strains from Cluster B were almost
identical to those of the type strains of S.bovis (99n9%)
and S.infantarius (99n9%), whereas the sequences of
the strains belonging to Cluster D were highly related
to that of the S.caprinus type strain (99n8 %). However,
from the phylogenetic tree shown in Fig. 1 it can be
seen clearly that the sodAint sequences of strains
belonging to Clusters B and D are not closely related to
those of S.bovis,S.infantarius,S.caprinus or to any of
1252 International Journal of Systematic and Evolutionary Microbiology 52

Dissection of S.bovis group using sodA sequences
the other type species studied here. These results
confirm our proposal that the sodA gene is a more
discriminative target sequence than the 16S rRNA
gene for differentiating closely related species belong-
ing to the genera Streptococcus and Enterococcus
(Poyart et al., 1998, 2000).
In conclusion, we have determined the sodAint se-
quences of 36 strains belonging to the S.bovis–
S.equinus group, including those for the type strains
of S.bovis,S.equinus,S.gallolyticus,S.caprinus,S.
infantarius,S.macedonicus and S.waius. The results
obtained from our analyses demonstrate that this
group comprises five different clusters that might
correspond to five distinct species. Three of these
species correspond to the previously characterized
species S.equinus,S.gallolyticus and S.infantarius.Of
the two remaining species, one comprised all of the
strains identified by phenotypic methods as belonging
to S.bovis biotype II.2. We propose the reassignment
of these strains to a novel species, S.pasteurianus. The
other species corresponds to strains previously identi-
fied as ‘S.infantarius subsp. coli’, for which the name S.
lutetiensis is proposed. This work demonstrates the
usefulness of a sodA-based database for the species
identification of related streptococcal isolates, and
suggests that sequence databases of defined DNA
targets, such as sodA, constitute a valuable alternative
approach for modern bacterial systematics. It also
confirms that the species S.equinus sensu stricto is
almost never isolated from human specimens and that
human clinical isolates are mostly composed of S.
gallolyticus,S.infantarius,S.pasteurianus and S.
lutetiensis.
Description of Streptococcus lutetiensis sp. nov.
Streptococcus lutetiensis (lu.tehti.en.sis. L. masc. n.
lutetia of Paris, where the species was characterized).
One of the strains of this species has been characterized
previously as ‘S.infantarius subsp. coli’ (Schlegel et al.,
2000). Cells are Gram-positive cocci that occur in pairs
or short chains. They are non-motile, non-sporulating,
catalase-negative and facultatively anaerobic. Most
strains show homogeneous growth in BHI and glucose
broths after 18 h incubation at 37 mC. Growth also
occurs in MRS broth without gas production. No
growth occurs in 6n5% NaCl broth. Colonies on blood
agar or nutrient agar are circular, smooth, entire and
non-pigmented. α-Haemolytic on blood agar. Charac-
teristics useful in the differentiation of S.lutetiensis
from its related streptococci are listed in Table 3. The
type strain of Streptococcus lutetiensis (NEM 782Tl
CIP 106849T) is a human isolate of unknown origin.
Three strains of this species were isolated from human
specimens (stool, cerebrospinal fluid and unknown
origin). Some other characteristics for S.lutetiensis can
be found in Schlegel et al. (2000).
Table 3. Tests useful in differentiating S. lutetiensis and
S. pasteurianus from major species belonging to the S.
bovis–S. equinus complex
.................................................................................................................................................
Species: 1, S.lutetiensis ;2,S.pasteurianus ;3,S.infantarius;
4, S.gallolyticus;5,S.macedonicus ;6,S.bovis;7,S.equinus.
The data presented here are based on the results obtained by
testing our strains and on data from Clarridge et al. (2001),
Devriese et al. (1998), Farrow et al. (1984), Nelms et al.
(1995), Osawa et al. (1995), Schlegel et al. (2000) and
Tsakalidou et al. (1998). The sodAint cluster refers to the
divisions shown in Fig. 1. j, More than 85 % of the strains
tested positive; k, more than 85 % of the strains tested
negative; v, variable test reaction. α-GAL, α-Galactosidase;
β-GAL, β-galactosidase; β-GLU, β-glucosidase ; β-GLC, β-
glucuronidase; β-MAN, β-mannosidase. None of the species
hydrolysed hippurate or urea. None of the species produced
arginine dihydrolase, alkaline phosphatase, pyrollidonyl
arylamidase, N-acetyl β-glucosamidase or glycyl tryptophan
arylamidase. All of the species tested produced acetoin, alanyl
phenylalanyl proline arylamidase and leucine aminopeptidase.
Acid was not produced from -arabinose, -arabitol,
cyclodextrin, ribose or sorbitol by any of the species tested.
Acid was produced from maltose, sucrose and methyl
β--glucopyranoside by all of the species.
Characteristics 1234567
sodAint cluster BDAEECC
Hydrolysis of:
Aesculin jj vjkjj
Production of:
α-GAL jjjjkjk
β-GAL kjkkkkk
β-GLU jj vkkj v
β-GLC kjkkkkk
β-MAN kjkkkkk
Acid from:
Glycogen kkjjkkk
Lactose jjjjjjk
-Mannitol kkkjkkk
Meleizitose kkkjkkk
Melibiose kjjkkkk
Pullulan kkjjkkk
Raffinose jj vjjkk
Tagatose kvkkkkk
Trehalose kjkjkkj
Starch jkjjjjk
Description of Streptococcus pasteurianus sp. nov.
Streptococcus pasteurianus (pas.teuhri.an.us. N.L. n.
pasteurianus of the Pasteur Institute, where the species
was characterized).
Strains of this species have been characterized pre-
viously as belonging to S.bovis biotype II.2 (Farrow
et al., 1984; Clarridge et al., 2001). Cells are Gram-
positive cocci that occur in pairs or short chains. They
http://ijs.sgmjournals.org 1253

C. Poyart, G. Quesne and P. Trieu-Cuot
are non-motile, non-sporulating, catalase-negative
and facultatively anaerobic. Most strains show homo-
geneous growth in BHI and glucose broths after 18 h
incubation at 37 mC. Growth also occurs in MRS broth
without gas production. No growth occurs in 6n5%
NaCl broth. Colonies on blood agar or nutrient agar
are circular, smooth, entire and non-pigmented. α-
Haemolytic on blood agar. Characteristics useful in
the differentiation of streptococci related to S.paster-
eurianus are listed in Table 3. The type strain of
Streptococcus pasteurianus (NEM 1202TlCIP
107122T) is a human isolate responsible for meningitis
and was isolated from the cerebrospinal fluid of an
infant. The five other strains of this species were
isolated from human specimen cultures (cerebrospinal
fluid, nl1; blood, nl3 ; urine, nl1). Some other
characteristics for S.pasteurianus can be found in
Clarridge et al. (2001) and Schlegel et al. (2000).
ACKNOWLEDGEMENTS
We thank C. Bizet for the gift of the streptococcal type
strains (CIP, Collection de l’Institut Pasteur, Institut Pas-
teur, 28 Rue du Docteur Roux, 75724 Paris Cedex 15,
France), and N. Fortineau (Laboratoire de Microbiologie,
Ho
#pital de Bice
#tre, 78 Avenue du Ge
!ne
!ral Leclerc, 94275 Le
Kremlin-Bice
#tre Cedex, France), R. Leclercq (Laboratoire
de Microbiologie, Centre Hospitalier Universitaire, Avenue
de la Co
#te Nacre, 14033 Caen Cedex, France) and F.
Vandenesch (Laboratoire de Microbiologie, Ho
#pital
Edouard Herriot, 5 Place d’Arsonval, 69394 Lyon Cedex 03,
France) for their gifts of clinical isolates. We also thank A.
Perrin for technical assistance in the hybridization experi-
ments, O. Gaillot and S. Nair for their critical reading of the
manuscript and P. Berche for his interest in this work. This
work was supported by the Pasteur Institute and by the
University of Paris V.
NOTE ADDED IN PROOF
During the preparation of this paper, the reclassification of
S.waius as S.macedonicus was proposed by Manachini et al.
(2002).
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