Streptococcus tigurinus sp. nov., isolated from blood of patients with endocarditis, meningitis and spondylodiscitis

Abstract

Four Gram-stain-positive, catalase-negative, coccus-shaped bacterial strains were isolated from multiple blood cultures of patients with endocarditis, meningitis and spondylodiscitis. The isolates were tentatively identified as viridans streptococcal species based on phenotypic characteristics. Comparative 16S rRNA gene sequencing studies showed that the organisms were members of the Streptococcus mitis group but did not correspond to any recognized species. The nearest phylogenetic relative was Streptococcus mitis ATCC 49456(T) with 98.6% sequence similarity. The representative strain AZ_3a(T) showed similarity values of less than 96.8%, 97.6%, 94.5% and 95.5% to the phylogenetically most closely related species by recA, rpoB, sodA and groEL gene sequence analysis, respectively. DNA-DNA hybridization analyses showed a low reassociation value of 32.2% between strain AZ_3a(T) and S. mitis DSM 12643(T). Reassociation values with other S. mitis group species ranged from 27.3% to 49.7%. The G+C content of the DNA is 40.0 mol%. Based on our biochemical and molecular analyses, the novel isolates represent a new species, for which the name Streptococcus tigurinus sp. nov. is proposed. The type strain is AZ_3a(T)( =CCOS 600(T) =DSM 24864(T)).

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Streptococcus tigurinus sp. nov., isolated from
blood of patients with endocarditis, meningitis and
spondylodiscitis
Andrea Zbinden,
1
Nicolas J. Mueller,
2
Philip E. Tarr,
3
Cathrin Spro¨er,
4
Peter M. Keller
1
3and Guido V. Bloemberg
1
Correspondence
Andrea Zbinden
azbinden@imm.uzh.ch
1
Institute of Medical Microbiology, University of Zurich, Gloriastrasse 30/32, CH-8006 Zu
¨rich,
Switzerland
2
Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich,
Ra¨mistrasse 100, CH-8091 Zu
¨rich, Switzerland
3
Infectious Diseases Service, Kantonsspital Bruderholz, University of Basel, Petersplatz 1,
CH-4003 Basel, Switzerland
4
DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7B,
D-38124 Braunschweig, Germany
Four Gram-stain-positive, catalase-negative, coccus-shaped bacterial strains were isolated from
multiple blood cultures of patients with endocarditis, meningitis and spondylodiscitis. The isolates
were tentatively identified as viridans streptococci on the basis of phenotypic characteristics.
Comparative 16S rRNA gene sequencing studies showed that the organisms were members of
the Streptococcus mitis group but did not correspond to any recognized species. The nearest
phylogenetic relative was S. mitis ATCC 49456
T
, with 98.6 % sequence similarity. The
representative strain AZ_3a
T
showed less than 96.8, 97.6, 94.5 and 95.5 % similarity to the
phylogenetically most closely related species by recA,rpoB,sodA and groEL gene sequence
analysis, respectively. DNA–DNA hybridization analyses showed a low reassociation value of
32.2 % between strain AZ_3a
T
and S. mitis DSM 12643
T
. Reassociation values with members of
other S. mitis group species ranged from 27.3 to 49.7 %. The G+C content of the DNA was
40.0 mol%. Based on our biochemical and molecular analyses, the isolates represent a novel
species, for which the name Streptococcus tigurinus sp. nov. is proposed. The type strain is
AZ_3a
T
(5CCOS 600
T
5DSM 24864
T
).
Accurate identification of bacteria within the Streptococcus
mitis group remains a challenge, in particular for Strep-
tococcus mitis,S. pneumoniae,S. pseudopneumoniae and S.
oralis. Conventional phenotypic methods are limited in
providing an accurate identification (Arbique et al., 2004),
and sequence analysis of the 59part of the 16S rRNA gene
is not sufficiently discriminative to differentiate these
species, because the sequence similarity is .99 % (Arbique
et al., 2004; Kawamura et al., 1995). Several other target
genes such as sodA (Kawamura et al., 1999; Poyart et al.,
1998), rpoB (Drancourt et al., 2004) and groEL (Glazunova
et al., 2009) have been investigated for species differenti-
ation, primarily using type strains, and we recently proposed
recA as an alternative target to differentiate S. pneumoniae
from other viridans streptococci (Zbinden et al., 2011).
Since commensal species such as S. oralis and S. mitis have
been recognized as important agents of endocarditis
(Douglas et al., 1993; Spellerberg & Brandt, 2011), accurate
identification of these bacteria is important.
Recently, we isolated a viridans streptococcal organism
from a 74-year-old patient with endocarditis that was
present in six out of six blood cultures and was designated
strain AZ_3a
T
. Molecular analyses by 16S rRNA gene
sequencing were performed for accurate identification. An
identical sequence was also detected in an aortic valve
specimen of the patient by direct 16S rRNA gene broad-
range PCR (Bosshard et al., 2003). BLAST analysis of the 16S
3Present address: Institute of Medical Microbiology, Friedrich-Schiller
University of Jena, PF 07737 Jena, Germany.
Abbreviation: MALDI-TOF, matrix-assisted laser desorption ionization-
time of flight.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA,
recA,rpoB,sodA and groEL gene sequences of strain AZ_3a
T
are
JN004270, JN004271, JQ085955, JQ085956 and JQ085957,
respectively.
Five supplementary figures are available with the online version of this
paper.
International Journal of Systematic and Evolutionary Microbiology (2012), 62, 2941–2945 DOI 10.1099/ijs.0.038299-0
038299 G2012 IUMS Printed in Great Britain 2941

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rRNA gene sequence of strain AZ_3a
T
was initially
performed using SmartGene software. BLAST searches to
reference sequences in the public databases revealed the
highest sequence similarity to the sequence of S. mitis ATCC
15914 (GenBank accession no. AY281076), with 99.9 %
identity; the next best reference sequence was that of the S.
mitis type strain, ATCC 49456
T
(AY485601), with 98.6 %
sequence identity. This relatively high sequence demarcation
of 1.3 % strongly suggested the recognition of a novel
streptococcal species and an erroneous assignment of strain
ATCC 15914, which was typed in 1977 based solely on
phenotypic characteristics (Facklam, 1977). Moreover, the
correct species assignment of strain ATCC 15914 was
questioned recently by several reports on the basis of
analyses of the housekeeping genes zwf and gki (Kiratisin
et al., 2005) and the 16S–23S rRNA intergenic spacer region
(Tung et al., 2006, 2007). A retrospective analysis of our
molecular database revealed three additional clinical strep-
tococcal isolates (AZ_4a, AZ_7a, AZ_10) that displayed the
closest 16S rRNA gene sequence similarity to strain ATCC
15914 in the public database. These three isolates were
derived from multiple blood cultures of patients with
endocarditis, meningitis and spondylodiscitis. Further
evaluation by conventional biochemical testing, molecular
analyses and DNA–DNA hybridization studies revealed that
the four new isolates and strain ATCC 15914 represent a
novel species of the S. mitis group.
The streptococcal strains ATCC 15914, AZ_3a
T
,AZ_4a,
AZ_7a and AZ_10 were grown on Columbia agar plates
containing 5 % defibrinated sheep blood (bioMe
´rieux) at
37 uC under aerobic conditions and in liquid culture using
brain heart infusion broth (Becton Dickinson). Type strains
S. pneumoniae DSM 20566
T
,S. mitis DSM 12643
T
,S. oralis
DSM 20627
T
,S. pseudopneumoniae CIP 108659
T
and
Streptococcus infantis CIP 105949
T
were obtained from the
DSMZ and the Institut Pasteur as indicated. Strain ATCC
15914 was purchased from the American Type Culture
Collection.
To determine the phylogenetic affinity of the isolates,
almost the entire 16S rRNA gene of each strain was
sequenced and subjected to a comparative analysis. A large
continuous fragment was obtained using universal primers
BAK11w (59-AGTTTGATCMTGGCTCAG-39; positions
10–27, Escherichia coli numbering) and Bact-1525a
(59-AAGGAGGTGATCCARCC-39; positions 1541–1525).
Cycling parameters included an initial denaturation for
5 min at 95 uC, 40 cycles of 1 min at 94 uC, 1 min at 48 uC
and 1 min at 72 uC, and a final extension for 10 min at
72 uC. The amplicons were sequenced bidirectionally with
an automatic DNA sequencer (ABI Prism 310 Genetic
Analyzer; Applied Biosystems). Multiple alignment of the
sequences was performed with the CLUSTAL V program
(Higgins et al., 1992) (MEGALIGN Lasergene version 7;
DNASTAR). Comparative 16S rRNA gene sequence analysis
revealed 99.8–100 % sequence similarity between isolates
AZ_3a
T
,AZ_4a, AZ_7a and AZ_10 and strain ATCC
15914, thereby demonstrating their high genealogical
relatedness. Sequences of the type strains of members of
the S. mitis group were retrieved from GenBank and the
neighbour-joining method (Saitou & Nei, 1987) was used
to reconstruct a phylogenetic tree. Strain AZ_3a
T
formed a
distinct branch within the S. mitis group (Fig. 1) and
revealed a clear affiliation to the genus Streptococcus (an
extended version of this tree is available in IJSEM Online as
Fig. S1). It is evident from Fig. 1 that strain AZ_3a
T
displays a phylogenetic affinity to a subcluster of species
consisting of S. mitis,S. pseudopneumoniae,S. pneumoniae,
S. oralis and S. infantis. Sequence similarity values were
calculated by using SmartGene software. Strain AZ_3a
T
displayed the highest sequence similarity to S. mitis ATCC
49456
T
(98.6 %); the next most closely related strains were
S. infantis ATCC 700779
T
(98.5 %), S. pseudopneumoniae
ATCC BAA-960
T
(98.3 %), S. pneumoniae ATCC 33400
T
(98.2 %) and S. oralis ATCC 35037
T
(98.1 %).
Additional gene sequence analyses were carried out in
order to analyse the phylogenetic affinities of the new
isolates and strain ATCC 15914 in more detail. A 313 bp
fragment of the recA gene was amplified with primers recA
2F and recA 5R for sequence analysis as described previously
(Zbinden et al., 2011). Isolates AZ_3a
T
,AZ_4a, AZ_7a and
AZ_10 and strain ATCC 15914 displayed an intraspecies
variability of 93.0–100 %. The highest recA sequence
similarity of strain AZ_3a
T
was observed with S. oralis
DSM 20627
T
(96.8 %). A phylogenetic tree (Fig. S2)
reconstructed by the neighbour-joining method with partial
sequences of the recA gene confirmed the phylogenetic
placement of the representative strain within the genus
Streptococcus.
S. sinensis HKU4T(AF432856)
S. oligofermentans 2-4T(AY099095)
S. cristatus ATCC 51100T(AY584476)
S. gordonii ATCC 10558T(AY485606)
S. sanguinis ATCC 10556T(DQ303192)
S. australis ATCC 700641T(AY485604)
S. parasanguinis ATCC 15912T(AY485605)
S. pneumoniae ATCC 33400T(AY485600)
S. pseudopneumoniae ATCC BAA-960T(AY612844)
S. mitis ATCC 49456T(AY485601)
S. oralis ATCC 35037T(AY485602)
S. infantis ATCC 700779T(AY485603)
S. tigurinus AZ_3aT(JN004270)
0.01
93.4
65.3
65.6
86.5
S. peroris GTC 848T(AB008314)
54.4
94.4
Fig. 1. Neighbour-joining phylogenetic tree
based on partial 16S rRNA gene sequences
(.1300 bp), showing relationships among
strain AZ_3a
T
(Streptococcus tigurinus sp.
nov.) and related species within the S. mitis
group. Bootstrap percentages (based on 1000
replications) .50 % are shown at branching
points. Published sequences used were from
the GenBank database. Bar, 0.01 substitu-
tions per nucleotide position. An extended
version of this tree is available as Fig. S1.
A. Zbinden and others
2942 International Journal of Systematic and Evolutionary Microbiology 62

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Sequence analysis of the rpoB,sodA and groEL genes has
also been demonstrated to be useful for phylogenetic
differentiation of streptococcal species (Drancourt et al.,
2004; Glazunova et al., 2009; Poyart et al., 1998). Partial
sequences of rpoB,sodA and groEL were obtained follow-
ing amplification using primers StreptoF and StreptoR
(Drancourt et al., 2004), d1 and d2 (Poyart et al., 1998) and
streptogroELd and streptogroELr (Glazunova et al., 2009),
respectively. Isolates AZ_3a
T
,AZ_4a, AZ_7a and AZ_10 and
strain ATCC 15914 shared 89.5–95.7 % sequence similarity
for the rpoB gene, 91.0–98.8 % for sodA and 93.2–95.3 % for
groEL. When comparing the rpoB,sodA and groEL gene
sequences of strain AZ_3a
T
with representative reference
sequences from species of the S. mitis group available in
GenBank, strain AZ_3a
T
formed a branch separate from
other S. mitis group species in phylogenetic trees inferred
from rpoB,sodA and groEL gene sequence comparisons (Figs
S3, S4 and S5). Strain AZ_3a
T
exhibited the highest sequence
similarity with the type strain of Streptococcus oligofermen-
tans (97.6, 94.5 and 95.5 %, respectively) based on rpoB,
sodA, and groEL. By analyses of housekeeping gene
sequences, we observed heterogeneity within the four new
isolates and strain ATCC 15914. Comparable intraspecies
variability for housekeeping genes (e.g. rpoB,gdh and recA)
has been reported for closely related S. mitis group species,
e.g. S. oralis and S. mitis (Hoshino et al., 2005; Kilian et al.,
2008; Nielsen et al., 2009; Sistek et al., 2012). This indicates a
limited potential of these genes for species assignment
within the S. mitis group. In contrast, analyses of the 16S
rRNA gene showed high similarity (99.8–100 %) among the
four new isolates (AZ_3a
T
,AZ_4a, AZ_7a and AZ_10) and
strain ATCC 15914.
The four new isolates and strain ATCC 15914 were
characterized phenotypically in detail. Strains were Gram-
stained and assessed for the presence of catalase. Haemolytic
reaction was determined on Columbia agar plates contain-
ing 5 % defibrinated sheep blood (bioMe
´rieux) incubated at
37 uC under aerobic conditions for 24 h. Growth was
determined at 25–45 uC as well as in brain heart infusion
broth containing 6.5% NaCl. Biochemical data were
obtained by using API 20 Strep, API 50 CH and Rapid ID
32 STREP kits (bioMe
´rieux). Identification was performed
according to the instructions of the manufacturer. API 50
CH strips using CHB/E suspension medium were read after
24 and 48 h of incubation at 37 uC. The isolates, including
strain ATCC 15914, exhibited almost identical biochemical
characteristics, except for the acidification of trehalose
(isolate AZ_3a
T
was negative). Phenotypic characteristics
that differentiate the proposed species from closely related
species are shown in Table 1.
Analysis of the new isolates and strain ATCC 15914 by the
VITEK 2 colorimetric card (bioMe
´rieux) revealed iden-
tification as S. mitis/S. oralis. In addition, analysis by
matrix-assisted laser desorption ionization-time of flight
mass spectrometry (MALDI-TOF MS) was performed with
a Microflex LT mass spectrometer (Bruker Daltonik) using
the MALDI Biotyper software package (version 3.0) with
reference database V.3.1.2.0 (Bruker Daltonik). Sample
preparation was done using the ethanol/formic acid
extraction protocol according to the manufacturer’s
instructions. For all isolates including strain ATCC
15914, MALDI-TOF MS analysis yielded scores of ¢2.2
with S. pneumoniae, suggesting identification at the species
level. However, accurate differentiation within the S. mitis
group by MALDI-TOF MS has been shown to be limited,
and such results should be interpreted with caution
(Ferroni et al., 2010; van Veen et al., 2010). The VITEK
and MALDI-TOF commercial identification systems might
be useful as a screening method for assignment of the
unknown organism to the S. mitis group, but not for
accurate species identification.
DNA–DNA hybridization studies were performed with
strain AZ_3a
T
and the type strains of its nearest
phylogenetic neighbours. Cells were disrupted by using a
French pressure cell (Thermo Spectronic) and genomic
DNA in the crude lysate was purified by chromatography
on hydroxyapatite as described by Cashion et al. (1977).
DNA–DNA hybridization was carried out by the
Identification Service of the DSMZ under optimal condi-
tions (26SSC at 66 uC) as described by De Ley et al.
(1970) under consideration of the modifications described
by Huß et al. (1983) by using a model Cary 100 Bio UV/Vis
spectrophotometer equipped with a Peltier-thermostatted
666 multicell changer and a temperature controller with an
Table 1. Biochemical characteristics of S. tigurinus sp. nov.
and the closest related species within the S. mitis group
Species: 1, S. tigurinus sp. nov. (results for isolates AZ_3a
T
,AZ_4a,
AZ_7a and AZ_10 and strain ATCC 15914); 2, S. mitis;3,S. oralis;4,
S. pneumoniae;5,S. pseudopneumoniae (results for strain CIP
108659
T
); 6, S. infantis;7,S. pyogenes. Data for S. tigurinus sp. nov.
and S. pseudopneumoniae were obtained in this study; data for S.
mitis,S. oralis,S. pneumoniae,S. infantis and S. pyogenes are from
Whiley & Hardie (2009). +,.85 % positive; d, different strains give
different reactions (16–84 % positive); 2, 0–15 % positive; ND,no
data available.
Characteristic 1 2345 67
Acid production from:
Glycogen 222+22d
Inulin 222+2dND
Melibiose 2dd+222
Raffinose +dd+222
Ribose 2dd22 2 2
Starch +2dd2ND ND
Trehalose d d d d 22+
Hydrolysis of arginine 2d2+22+
Production of:
N-Acetyl-b-glucosaminidase 2++ d2d2
Alkaline phosphatase 2d+2+2+
a-D-Galactosidase 2dd+222
b-D-Galactosidase +d+++ + 2
Glycyl-tryptophan arylamidase 2d++ 222
Streptococcus tigurinus sp. nov.
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in situ temperature probe (Varian). The DNA–DNA
hybridization studies (performed in duplicate) showed that
the strain that was most closely related to strain AZ_3a
T
was
S. oralis DSM 20627
T
, with 49.7±2.2 % relatedness. The
next most closely related strains displayed relatedness values
of 40.9±4.2 % (S. pneumoniae DSM 20566
T
), 35.4±4.6 %
(S. pseudopneumoniae CIP 108659
T
), 32.2±4.2 % (S. mitis
DSM 12643
T
) and 27.3±4.0 % (S. infantis CIP 105949
T
).
These reassociation values were well below 70%, indicating
that strain AZ_3a
T
is distinct and separate from the closest
related species (Wayne et al., 1987). The genomic DNA
G+C content of strain AZ_3a
T
was determined by using the
HPLC method of Mesbah et al. (1989) as 40.0 mol%.
On the basis of the hybridization results and the above-
described genetic and phenotypic analyses, we propose the
description of a novel species, Streptococcus tigurinus sp.
nov., with the type strain AZ_3a
T
, and reassignment of
strain ATCC 15914 to this species. Analysis by 16S rRNA
gene sequencing facilitates accurate identification of S.
tigurinus sp. nov. In view of its association with serious
clinical manifestations, e.g. endocarditis, meningitis and
spondylodiscitis, further epidemiological analyses are
needed in order to describe in detail the natural pathogenic
potential of this novel species.
Description of Streptococcus tigurinus sp. nov.
Streptococcus tigurinus (ti.gu.ri9nus. L. masc. adj. tigurinus
of or pertaining to Tigurum, a district in Helvetia, the
modern Zurich, the region where the bacterial species was
first recognized).
Gram-stain-positive, non-motile, non-spore-forming cocci,
0.5–1.0 mm in diameter, arranged in chains. Surface colonies
on sheep blood agar are circular, smooth, white to greyish,
convex, a-haemolytic and 0.5–1.0 mm in diameter after
24 h of incubation at 37 uC under an aerobic atmosphere.
Facultatively anaerobic; no enhancement of growth in 5 %
CO
2
. Growth occurs at 25–42 uC but not in 6.5 % NaCl
broth. Catalase-negative. Produces leucine aminopeptidase
and alanyl-phenylalanyl-proline arylamidase but not a-D-
galactosidase, alkaline phosphatase, pyrrolidonyl arylami-
dase, b-glucuronidase, b-glucosidase, pyroglutamic acid
arylamidase, b-mannosidase, urease, glycyl-tryptophan ary-
lamidase or N-acetyl-b-glucosaminidase. Production of b-D-
galactosidase depends on the substrate used [either resorufin
b-D-galactopyranoside (positive) or 2-naphthyl b-D-galac-
topyranoside (negative)]. Fermentation of starch, pullulan,
lactose, D-galactose, D-glucose, D-fructose, D-mannose,
maltose, raffinose and sucrose is observed, but D-ribose,
arabinose, D-mannitol, D-sorbitol, a-cyclodextrin, arabitol,
glycogen, melibiose, melezitose, methyl b-D-glucopyrano-
side, D-tagatose and inulin are not fermented. Fermentation
of trehalose is variable (positive for most strains; type strain
AZ_3a
T
negative). L-Arginine, hippurate and aesculin are
not hydrolysed. The Voges–Proskauer reaction is negative.
The type strain is AZ_3a
T
(5CCOS 600
T
5DSM 24864
T
),
isolated from human blood. The DNA G+C content of the
type strain AZ_3a
T
is 40.0 mol%. Additional members of
the species are isolates AZ_4a, AZ_7a and AZ_10 and strain
ATCC 15914 (originally identified as S. mitis).
Acknowledgements
The study was supported by the University of Zurich. We thank the
laboratory technicians and B. Schulthess for their dedicated help. We
thank G. Eich, S. Christen, T. Bruderer, P. Schumann, E. Acampora
and R. Zbinden for substantial assistance.
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