J
OURNAL OFC
LINICALM
ICROBIOLOGY, Feb. 2004, p. 497–504
Vol. 42, No. 2
0095-1137/04/$08.00
⫹
0 DOI: 10.1128/JCM.42.2.497–504.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
rpoB
Gene Sequence-Based Identification of Aerobic Gram-Positive
Cocci of the Genera
Streptococcus,
Enterococcus,
Gemella,
Abiotrophia, and
Granulicatella
Michel Drancourt, Ve´ronique Roux, Pierre-Edouard Fournier, and Didier Raoult*
Unite´ des Rickettsies, IFR 48, Faculte´ de Me´decine, Universite´ de la Me´diterrane´e, Marseille, France
Received 28 August 2003/Returned for modification 12 October 2003/Accepted 27 October 2003
We developed a new molecular tool based on
rpoB
gene (encoding the beta subunit of RNA polymerase)
sequencing to identify streptococci. We first sequenced the complete
rpoB
gene for
Streptococcus anginosus,
S.
equinus, and
Abiotrophia defectiva. Sequences were aligned with these of
S. pyogenes,
S. agalactiae, and
S.
pneumoniae
available in GenBank. Using an in-house analysis program (SVARAP), we identified a 740-bp
variable region surrounded by conserved, 20-bp zones and, by using these conserved zones as PCR primer
targets, we amplified and sequenced this variable region in an additional 30
Streptococcus,
Enterococcus,
Gemella,
Granulicatella, and
Abiotrophia
species. This region exhibited 71.2 to 99.3% interspecies homology. We
therefore applied our identification system by PCR amplification and sequencing to a collection of 102
streptococci and 60 bacterial isolates belonging to other genera. Amplicons were obtained in streptococci and
Bacillus cereus, and sequencing allowed us to make a correct identification of streptococci. Molecular signatures
were determined for the discrimination of closely related species within the
S. pneumoniae-S. oralis-S. mitis
group and the
S. agalactiae-S. difficile
group. These signatures allowed us to design a
S. pneumoniae-specific
PCR and sequencing primer pair.
Aerobic, gram-positive, catalase-negative cocci were initially
regarded as forming an unique phylum of bacteria roughly
corresponding to the genus
Streptococcus
(41). Broad changes
in the classification of the streptococci have resulted from
molecular taxonomic studies of the genus
Streptococcus
. The
enterococci, previously considered group D streptococci, now
reside in their own genus,
Enterococcus
(44). A new genus,
Abiotrophia
, has been proposed to accommodate nutritionally
variant streptococci (21). Finally, Collins and Lawson have
proposed that three species of the genus
Abiotrophia
be
reclas-sified into a new genus,
Granulicatella
(9).
These new genera accommodate bacterial isolates recovered
from environmental and clinical sources (41). In humans,
streptococci are responsible for a wide range of manifestations,
including both invasive and toxin-related manifestations such
as scarlet fever (1).
Streptococcus agalactiae
is the leading cause
of neonatal disease, requiring urgent diagnosis in pregnant
women (31).
S. pneumoniae
is the bacterial species most
fre-quently isolated in cerebrospinal fluid from individuals with
adult meningitis (27). It requires rapid detection, even in the
case of culture-negative meningitis due to antibiotic treatment.
Also, microorganisms of these five genera remain the leading
cause of infective endocarditis worldwide, a life-threatening
condition requiring rapid antibiotic treatment based on
effec-tive identification of the causing species. Also, because some
species are fastidious and highly susceptible to antibiotics,
these are responsible for blood culture-negative endocarditis.
Emerging taxons of streptococci have been described in this
situation such as
S. sinensis
(51),
S. pasteurianus
(37),
S.
lute-tiensis
(37),
Enterococcus hirae
(38), and
Granulicatella elegans
(7). Recent reappraisal of these taxons demonstrated that
S.
lutetiensis
was a genotype of
S. infantarius
and that
S.
pasteur-ianus
was a subspecies of
S. gallolyticus
(45). In these situations
also, molecular detection and identification of the causative
agent should be done even after antibiotic treatment resulted
in culture-negative endocarditis.
In clinical laboratories, the current means of identification
of streptococci and related genera rely on phenotypic tests,
such as those developed into the API ID 32 Strep system (Bio
Me´rieux, la Balme les Grottes, France). However, the
poten-tial problems inherent in the use of phenotypic tests are that
not all strains within a given species may be positive for a
common trait (3, 24) and that the same strain may exhibit
biochemical variability (19, 48). Consequently, the routine
technique based on phenotypic tests does not allow for an
unequivocal identification of certain streptococcal species, in
particular those belonging to the
S. milleri
, the
S. mutans
and
the
S. mitis
groups (3, 14, 24).
Nucleic acid probes have been developed for the
identifica-tion of isolates of group A and B streptococci (10, 18). For the
identification of
S. pneumoniae
, a commercial probe has been
increasingly used (11, 16), and different PCR-based methods
detected genes for pneumococcal toxins or other virulence
factors, such as the pneumolysin (
ply
) (42) and the major
autolysin (
lytA
) not normally present in other alpha-hemolytic
streptococci (20, 33, 43). Also, PCR-based techniques targeted
the streptococcal 16S-23S rRNA spacer region (15), the C
protein gene of
S. agalactiae
(5, 29), the
groESL
genes of
viridans group streptococci (49), and
sodA
gene encoding the
manganese-dependent superoxide dismutase in several
Strep-tococcus
and
Enterococcus
species (34, 35, 36) by using two
different pairs of primers. However, no molecular tool
com-* Corresponding author. Mailing address: Unite´ des Rickettsies,
IFR 48, Faculte´ de Me´decine, Universite´ de la Me´diterrane´e,
Mar-seille, France. Phone: 04-91-38-55-17. Fax: 04-91-38-77-72. E-mail:
[email protected].
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prised these five genera at one time, with the exception of 16S
rRNA gene, which did not discriminate all species (4).
rpoB
, the gene encoding the highly conserved subunit of the
bacterial RNA polymerase, has previously been demonstrated
to be a suitable target on which to base the identification of
enteric bacteria (30), spirochetes (28, 41), bartonellas (40),
rickettsias (13), legionellae (26), mycobacteria (17, 25),
staph-ylococci (12),
Bacillus
spp. (39), and ehrlichiae (47). The gene
has been shown to be more discriminative than the 16S rRNA
gene (30) for identifying enteric bacteria. In this report, we
describe the molecular identification of aerobic, gram-positive
catalase-negative species of the genera
Streptococcus
,
Entero-coccus
,
Gemella
,
Abiotrophia
, and
Granulicatella
by using a
single specific primer pair for PCR and sequencing method
based on the sequence of the
rpoB
gene.
MATERIALS AND METHODS
Bacterial strains.The 33 streptococcal type strains used in the present study
are listed in Table 1. They were grown on blood agar at 37°C under a 5% CO2
atmosphere. All isolates were streaked on blood agar plates to determine the purity of each of the cultures by macroscopic examination of colonies and microscopic examination of Gram-stained preparations. Colonies were scraped from the plates and boiled for 15 min in Chelex 100 (46) and 1% sodium docecyl sulfate solution (Bio-Rad Laboratories, Hercules, Calif.) before genomic DNA was extracted and purified with QIAmp DNA minikits (Qiagen GmbH, Hilden,
Germany). The 162 clinical isolates used in blind identification testing (see below) are listed in Table 2.
Determination of the completerpoBsequence inS. equinus,S. anginosus, and
Abiotrophia defectiva.ConsensusrpoBPCR primers were designed after the
alignment ofrpoBgenes ofS. pyogenes(GenBank accession number AE006480),
S. pneumoniae(GenBank accession number AE008542), andBacillus subtilis
(GenBank accession number L43593) and numbered on the basis of theS.
agalactiae rpoBsequence (Table 3). Primer pair 31F (5⬘-GCCTTAGGACCTG
GTGGTTT-3⬘)-830R (5⬘-GTTGTAACCTTCCAWGTCAT-3⬘) was used to
am-plify arpoBgene fragment inS. equinus,S. anginosus, andA. defectiva.These
species were chosen as representatives of the major phyla based on analysis of the phylogenetic tree derived from the 16S rRNA sequencing. Additional oligo-nucleotides were selected on the basis of data obtained from ongoing base sequence determination (Table 2). The forward primers 371F, 730F, and 1848F combined with the reverse primers 585R, 1252R, 2057R, and 2215R were used
to amplify and sequence additional portions of therpoBgene in these three
species. All PCR mixtures contained 2.5⫻102U ofTaqpolymerase/l, 1⫻Taq
buffer, 1.8 mM MgCl2(Gibco-BRL/Life Technologies, Cergy Pontoise, France),
200M concentrations of dATP, dTTP, dGTP, and dCTP (Boehringer
Man-heim GmbH, Hilden, Germany), and 0.2M concentrations of each primer
(Eurogentec, Serraing, Belgium). PCR mixtures were subjected to 35 cycles of denaturation at 94°C for 30 s, primer annealing at 52°C for 30 s, and de novo DNA extension at 72°C for 60 s. Every amplification program began with a denaturation step of 95°C for 2 min and ended with a final elongation step of 72°C for 5 min. Amplicons were purified for sequencing by using a QIAquick spin PCR purification kit (Qiagen) according to the protocol of the supplier. The
sequences of the 3⬘and 5⬘extremities were determined by using the universal
Genomic Walker kit according to the manufacturer’s instructions (Clontech, Palo Alto, Calif.), incorporating primers 520R and 2881F and primers 1000R and
3000F forE. faecalis.
Sequence variability ofrpoBinStreptococcusspp. and related genera.
Inter-speciesrpoBgene sequence variability was analyzed by using the in-house
pro-gram sequence variability analysis propro-gram (SVARAP), which uses the Excel
program to simultaneously process sets of up to 100 sequences of⬍4,000
nu-cleotides and allows comparison of data from two sets of sequences. Successive site-by-site analysis and successive window analysis of 60 nucleotide sites were used to reveal regions with particular patterns of variability. We tabulated site variability as the proportion of sequences that differ from the consensus
se-quence at a given site. Variability was calculated as follows: 100⫺(maximum
value of frequency for each of the four nucleotides at a given position). Our program requires nucleotide sequence alignment format as input and produces a numerical and graphical portrayal of variability as output. This program was
applied to a file of eightrpoBcomplete sequences including the three complete
sequences determined in the present study (S. anginosus,S. equinus, andA.
defectiva) and sequences published in GenBank forS. pneumoniae(AE008542
and AE007486), S. agalactiae(AL766844 and AE014199), and S. pyogenes
(AE006480). After sequence alignment with CLUSTAL X, v.1.8. Aligned se-quences were copied, pasted into our program, and then automatically pro-cessed. Each nucleotide for each sequence was automatically assigned to a different cell in order to align nucleotides at a given position in the same column. The program then calculated the consensus nucleotide (defined as the most frequent nucleotide at a each site in the set of sequences), the absolute numbers of each of four nucleotides (G, A, C, and T), deletions, or insertions and their frequency (as a percentage). All of these data were processed for a window of 60 nucleotides to calculate the median, as well as the mean highest and lowest variabilities with the standard deviations, and the results were plotted within graphical windows. The program permitted the determination of a 740-bp vari-able region surrounded by two consensus regions, and this varivari-able region was
further tested for the constitution of anrpoB-streptococcus database.
Streptococcusand related genus partialrpoBsequence database.Partial re-verse and forward (bidirectional) sequencing of a 740-bp fragment was obtained
by using internal primers at positions 2333 (Strepto F [5⬘-AARYTIGGMCCT
GAAGAAAT-3⬘]) and 3073 (Strepto R [5⬘-TGIARTTTRTCATCAACCATGT
G-3⬘]). Sequencing reactions were carried out with the reagents of the ABI Prism
dRhodamine dye terminator cycle sequencing ready reaction kit (Perkin-Elmer Applied Biosystems, Foster City, Calif.) according to the manufacturer’s instruc-tions and with the following program: 30 cycles of denaturation at 94°C for 10 s, primer annealing at 50°C for 10 s, and extension at 60°C for 2 min. Products of sequencing reactions were separated by electrophoresis on a 0.2-mm 6% poly-acrylamide denaturing gel and recorded with an ABI Prism 377 DNA sequencer (Perkin-Elmer Applied Biosystems) according to the standard protocol of the
[image:2.603.43.284.88.418.2]supplier. This protocol was applied to the collection of 33Streptococcusand
TABLE 1. List of 33 streptococcus species investigated by
rpoB
sequencing
Species CIP straina GenBank
accession no.
Abiotrophia defectiva
103242
TAF535173
Enterococcus avium
103019
TAF535192
Enterococcus casselliflavus
103018
TAF535174
Enterococcus faecalis
103015
TAF535178
Enterococcus faecium
103014
TAF535176
Enterococcus gallinarum
103013
TAF535177
Enterococcus saccharolyticus
103246
TAF535175
Gemella haemolysans
101126
TAF535179
Gemella morbillorum
81.10
TAF535180
Granulicatella adjacens
103243
TAF535172
Streptococcus acidominimus
82.4
TAF535181
Streptococcus agalactiae
103227
TAF535182
Streptococcus anginosus
103244
TAF535183
Streptococcus bovis
102302
TAF535189
Streptococcus constellatus
103247
TAF535184
Streptococcus difficile
103768
TAF535191
Streptococcus dysgalactiae
102914
TAF535185
Streptococcus equi
102910
TAF535186
Streptococcus gallolyticus
105428
TAY315154
Streptococcus equinus
102504
TAF535187
Streptococcus infantarius
103233
TAY315155
Streptococcus intermedius
103248
TAF535190
Streptococcus lutetientis
106849
TAY315158
Streptococcus macedonicus
105683
TAY315156
Streptococcus mitis
103335
TAF535188
Streptococcus mutans
103220
TAF535167
Streptococcus oralis
102922
TAF535168
Streptococcus pasteurianus
107122
TAY315157
Streptococcus pneumoniae
10291
TAE008542
Streptococcus pyogenes
56.41
TAE006480
Streptococcus salivarius
102503
TAF535169
Streptococcus sanguinis
55.128
TAF535170
Streptococcus suis
103217
TAF535171
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related genus species listed in Table 1 in order to set up a reference partialrpoB gene database.
Molecular signatures in closely relatedStreptococcusspecies.PartialrpoB sequences of closely related species exhibiting ambiguous phenotypic and mo-lecular identifications were aligned for the search of positions that distinguished
related species (molecular signatures) (Fig. 1). For that purpose, we alignedrpoB
partial sequences determined in 10S. pneumoniaeisolates (including two
se-quences from GenBank, accession numbers AE008542 and AE007486) with that ofS. mitisandS. oralis. Based on these signatures, we designed the primer pair
rpoBpneumoF (3⬘-TGTTAACATGTTGGTTCGTGTT-5⬘) and rpoBpneumoR
(3⬘-CATCAAAGACTGGTGTCGCA-5⬘) for the specific amplification and
se-quencing ofS. pneumoniae rpoB. These primers were included in a PCR (under
the conditions described above except for a hybridization temperature of 56°C)
incorporating tenS. pneumoniaeisolates, fiveS. mitisisolates, and fiveS. oralis
isolates. Likewise, we alignedrpoBsequences determined in tenS. agalactiae
isolates (including two sequences from GenBank, accession numbers AL766844
and AE014199) withS. difficile. Combinations of base positions unique toS.
pneumoniaeandS. agalactiaewere defined as molecular signatures for these species.
[image:3.603.46.536.87.364.2]rpoBsequence-based identification blind testing.TherpoB-based system we developed to identify streptococci was applied to a collection of 162 clinical isolates in order to assess its specificity (Table 2). This collection included 102 isolates of streptococci and 60 isolates belonging to other bacterial genera, including species responsible for endocarditis. After the isolates were coded,
TABLE 2. List of 162 bacterial clinical isolates used for blind identification testing by the
rpoB
gene sequence-based
method described in the text
Species No. of isolates Species No. of isolates
Abiotrophia defectiva
... 2
Acinetobacter baumanii
... 1
Actinobacillus actinomycetemcomitans
... 1
Actinomyces
spp. ... 3
Aeromonas hydrophila
... 1
Arcanobacterium haemolyticum
... 1
Aspergillus fumigatus
... 1
Bacillus cereus
... 3
Borrelia burgdorferi
... 1
Campylobacter fetus
... 1
Candida albicans
... 2
Clostridium
spp. ... 3
Corynebacterium
spp. ... 5
Coxiella burnetii
... 1
Escherichia coli
... 2
Eikenella corrodens
... 1
Enterobacter agglomerans
... 3
Enterococcus casselliflavus
... 1
Enterococcus faecalis
... 12
Enterococcus faecium
... 11
Enterococcus gallinarum
... 2
Gemella haemolyticus
... 3
Gemella morbillorum
... 1
Granulicatella adjacens
... 1
Haemophilus influenza
... 1
Klebsiella pneumoniae
... 1
Kluyvera ascorbata
... 1
Lactobacillus
spp. ... 2
Legionella pneumophila
... 1
Leuconostoc lactis
... 1
Moraxella lacunata
... 1
Mycobacterium
spp. ... 5
Mycoplasma pneumoniae
... 1
Norcardia asteroides
... 1
Neisseria meningitidis
... 1
Pasteurella multocida
... 1
Pediococcus acidilactici
... 1
Peptostreptococcus magnus
... 1
Propionibacterium acnes
... 3
Serratia marcescens
... 1
Staphylococcus
spp. ... 5
Streptococcus agalactiae
... 7
Streptococcus anginosus
... 5
Streptococcus bovis
... 2
Streptococcus constellatus
... 1
Steptococcus equinus
... 1
Streptococcus gordonii
... 1
Streptococcus infantarius
... 1
Streptococcus intermedius
... 4
Streptococcus mitis
... 8
Streptococcus oralis
... 7
Streptococcus pneumoniae
... 12
Streptococcus salivarius
... 1
Streptococcus sanguis
... 2
Turicella otitidis
... 1
TABLE 3. Primers used to sequence the entire
rpoB
gene in
S. anginosus
,
S. equinus
, and
A. defectiva
Sequence (5⬘-3⬘) Primer Tm(°C)
Forward Reverse
GCCTTAGGACCTGGTGGTTT
31F
58
CGTTGCATGTTGGCACCCAT
503R
58
GGACACATACGACCATAGTG
116R
56
AGACGGACCTTCTATGGAAAA
StrpoB 748F
56
GTTGTAACCTTCCCAWGTC AT
830R
56
AACCAATTCCGYATYGGTYT
1252F
53
GTCTTCWTGGGYGATTTCCC
2215F
57
ACCGTGGiGCWTGGTTRGAAT
205F
56
AGTGCCCAAACCTCCATCTC
730F
56
AGTGGGTTTAACATGATGTC
371F
50
CTCCAAGTGAACAGATGTGTA
585R
56
AACCAAGCYCCACGGTTAGGRAT
GW520R
64
ATGTTGAACCCACTiGGGGTGCCAT
GW2881F
64
TGTArTTTrTCATCAACCATGTG
Strepto R
52
AARYTiGGMCCTGAAGAAAT
Strepto F
52
CTTTACGATGGACGTACAGGTGAACCATTT
3000F
66
GCTGTCGTCTGGATAGCCGTTACCAAT
1000R
67
V
OL. 42, 2004
rpoB
GENE SEQUENCE-BASED IDENTIFICATION OF COCCI
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[image:3.603.41.541.536.725.2]TABLE
4.
Percentage
of
partial
rpoB
gene
sequence
similarity
in
33
Streptococcus
,
Enterococcus
,
Gemella
,
Abiotrophia
,and
Granulicatella
spp.
Species % rpoB gene sequence similarity awith: S.gallolyticus S.macedonicus S.pasteurianus S.anginosus S.intermedius S.constellatus S.lutetientis S.bovis S.equinus S.salivarius S.oralis S.pneumoniae S.dysgalactiae S.pyogenes G.morbillorum G.haemolysans S.infantarius S.mitis S.acidominimus S.suis S.sanguinis S.mutans S.agalactiae S.equi S.dif ficile E.faecalis E.avium E.sacharolyticus E.gallinarum E.casselli E.faecium G.adjacens S. gallolyticus 100.0 95.4 92.8 73.5 72.7 73.0 85.2 79.2 78.9 76.6 73.5 73.6 74.3 75.3 74.0 73.8 88.9 74.1 70.9 71.4 71.4 70.7 72.5 71.3 63.5 77.6 59.6 71.4 73.4 75.3 77.9 76 .0 S. macedonicus 100.0 96.0 76.3 75.5 75.9 88.5 82.3 82.3 79.7 76.3 76.8 77.5 78.5 71.0 70.7 90.7 76.9 74.0 74.5 74.6 73.2 75.6 73.9 66.1 74.6 62.2 75.0 75.0 73.0 74.4 73.2 S. pasteurianus 100.0 75.5 74.6 74.9 88.8 81.5 81.2 78.4 75.5 75.7 16.1 77.0 69.2 69.0 88.7 75.9 72.0 73.2 73.0 72.7 74.8 72.7 64.6 72.5 62.8 72.3 72.7 70.8 72.9 70.9 S. anginosus 100.0 93.3 92.0 79.1 66.1 85.9 86.2 83.4 83.3 85.4 85.2 62.7 62.8 75.2 83.8 82.8 83.6 82.1 83.0 84.6 83.0 75.9 66.0 69.7 63.4 67.6 63.7 63.8 63.0 S. intermedius 100.0 91.5 78.5 85.4 85.2 86.7 83.4 83.9 84.3 84.6 60.9 61.0 74.9 83.0 82.1 83.1 81.0 82.6 84.3 82.5 76.1 66.0 69.8 63.1 67.5 63.8 64.8 63.1 S. constellatus 100.0 77.8 84.8 84.6 86.1 84.6 83.8 84.1 84.1 61.3 61.4 74.2 84.4 81.8 84.4 83.4 81.1 83.0 82.5 73.9 64.8 69.3 62.6 66.0 62.0 64.2 63.6 S. lutetientis 100.0 90.3 89.4 83.8 81.0 81.0 80.9 81.2 69.1 69.0 93.5 80.4 77.2 78.9 76.8 77.4 79.7 76.8 70.0 72.0 66.8 71.4 72.8 69.3 70.4 70.0 S. bovis 100.0 98.7 91.6 88.4 88.5 89.0 89.2 64.4 64.5 85.9 88.2 84.1 85.9 83.6 84.3 87.9 84.3 76.9 67.8 71.1 66.3 67.5 64.5 65.6 65.6 S. equinus 100.0 90.3 87.2 88.0 88.0 87.9 64.5 64.6 85.1 87.2 84.6 85.7 84.1 84.3 87.5 83.9 77.0 67.7 70.7 66.2 67.5 64.6 65.2 65.8 S. salivarius 100.0 89.5 89.0 89.2 89.5 63.7 63.8 79.8 88.4 86.4 88.4 86.2 83.9 87.9 86.2 75.6 67.0 70.0 64.9 66.9 63.4 65.1 65.2 S. oralis 100.0 93.6 86.7 86.4 63.0 63.1 77.2 93.6 84.8 87.2 88.0 84.1 85.2 83.1 76.4 65.7 69.2 63.0 65.5 64.1 64.2 63.6 S. pneumoniae 100.0 85.6 85.6 63.8 63.9 77.2 94.4 84.9 87.4 86.6 83.4 85.4 82.6 77.0 66.0 70.7 63.2 65.5 64.6 64.5 63.6 S. dysgalactiae 100.0 96.4 62.9 62.9 77.0 85.2 85.1 86.4 84.4 85.2 86.9 86.7 75.9 66.6 71.1 64.4 66.6 63.7 63.8 63.8 S. pyogenes 100.0 62.9 62.9 77.3 84.8 84.4 85.4 83.8 84.8 86.2 86.4 75.7 67.1 70.7 64.2 67.1 63.5 63.5 63.3 G. morbillorum 100.0 99.4 71.1 63.1 62.6 62.4 60.9 61.8 63.7 60.9 63.4 75.8 58.9 75.5 71.8 74.8 75.7 75.8 G. haemolysans 100.0 71.1 63.2 62.7 62.5 61.0 61.8 63.8 61.0 63.5 75.9 59.0 75.6 71.8 74.7 75.5 75.5 S. infantarius 100.0 76.6 74.2 75.5 73.5 73.0 76.2 73.5 67.0 74.9 63.1 74.2 75.3 72.8 74.0 73.3 S. mitis 100.0 84.1 85.6 85.4 83.1 84.4 81.5 76.4 65.9 68.7 62.8 65.8 64.1 64.4 65.0 S. acidominimus 100.0 90.2 84.8 84.4 84.1 85.4 75.4 64.5 68.4 63.4 65.8 63.3 63.0 63.6 S. suis 100.0 86.4 85.1 85.7 83.1 75.9 66.4 70.3 64.4 65.6 63.7 65.1 64.7 S. sanguinis 100.0 81.6 82.0 83.8 74.8 63.7 67.9 62.7 65.0 62.7 63.5 61.8 S. mutans 100.0 85.9 85.9 74.9 64.9 69.3 63.2 65.9 62.9 64.4 63.7 S. agalactiae 100.0 85.7 76.4 66.4 71.3 64.8 67.2 64.1 64.9 64.8 S. equi 100.0 74.1 64.0 68.4 63.2 65.3 62.2 63.4 62.6 S. dif ficile 100.0 71.7 76.2 70.6 79.1 83.9 71.1 68.0 E. faecalis 100.0 66.7 84.3 80.2 83.8 84.2 81.9 E. avium 100.0 67.1 69.4 64.4 65.2 63.7 E. sacharolyticus 100.0 79.6 83.4 85.8 81.1 E. gallinarum 100.0 86.1 80.6 76.6 E. casselli 100.0 85.3 79.1 E. faecium 100.0 79.6 G. adjacens 100.0 A.defectiva aFull
genus and species names can be found in Table 2.
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extraction of bacterial DNA and PCRs incorporating primer pair Strepto F-Strepto R were performed as described above. The presence of 740-bp amplicons was revealed by 1% agarose gel electrophoresis, and amplicons were sequenced by using the sequencing primers described above. Sequences were aligned by
using FASTA with the streptococcusrpoBdatabase in Infobiogen (http://www
.infobiogen.fr/services/analyseq/cgi-bin/fasta㛭in.pl), and identification was
as-sessed by⬎97% similarity with one of the database sequence.
Nucleotide sequence accession numbers.The GenBank accession numbers of
the streptococcalrpoBsequences determined in the present study are AF535167
for theS. mutanscompleterpoBsequence, AF535187 for theS. equinuscomplete
rpoB sequence, AF535183 for the S. anginosus complete rpoB sequence,
AF535173 for theA. defectivacompleterpoBsequence, AF535168 for theS.
oralispartialrpoB sequence, AF535169 for theS. salivariuspartialrpoB
se-quence, AF535170 for theS. sanguinispartialrpoBsequence, AF535171 for the
S. suispartialrpoBsequence, AF535181 for theS. acidominimuspartialrpoB
sequence, AF535182 for theS. agalactiaepartialrpoBsequence, AF535184 for
theS. constellatuspartialrpoBsequence, AF535191 for theS. difficilispartial
rpoBsequence, AY315158 for theS. lutetiensispartialrpoBsequence, AY315157
for theS. pasteurianuspartialrpoBsequence, AY315156 for theS. macedonicus
partialrpoBsequence, AY315154 for theS. gallolyticuspartialrpoBsequence,
AY315155 for theS. infantariuspartialrpoBsequence, AF535185 for theS.
dysgalactiaepartialrpoBsequence, AF535186 for theS. equipartialrpoB
se-quence, AF535190 for theS. intermediuspartialrpoBsequence, AF535188 for
theS. mitispartialrpoBsequence, AF535189 for theS. bovispartialrpoB
se-quence, AF535192 for theE. aviumpartialrpoBsequence, AF535174 for theE.
casselliflavuspartialrpoBsequence, AF535178 for theE. faecalispartialrpoB
sequence, AF535176 for theE. faeciumpartialrpoBsequence, AF535177 for the
E. gallinarumpartialrpoBsequence, AF535175 for theE. saccharolyticuspartial
rpoB sequence, AF535179 for the G. haemolysans partial rpoB sequence,
AF535180 for theG. morbillorumpartialrpoBsequence, and AF535172 for the
G. adjacenspartialrpoBsequence.
RESULTS
Determination of
rpoB
sequences in
Streptococcus
and
re-lated genus species and construction of a partial
rpoB
se-quence database.
Consensus primers 31F and 830R permitted
the amplification of an 800-bp
rpoB
fragment in
S. anginosus
,
S.
equinus
, and
A. defectiva
, and additional consensus primers
pairs allowed us to determine the almost the entire
rpoB
se-quence in these three species: primers 1252F, 2057F, and
2215F combined with primers 116R and 503R allowed us to
further sequence the 3
⬘
extremity of the gene in the three
species, whereas combining primer 7487F with 585R, 371R,
and 730R allowed us to further sequence the 5
⬘
extremity. Both
3
⬘
and 5
⬘
extremities were obtained by using the genome
walker kit incorporating primer GW520R and primer
GW2881F, respectively. Overall, a complete 3,567-bp
se-quence was determined from the ATG start codon to the TGA
stop codon in
S. anginosus
, a 3,573-bp sequence was
deter-FIG. 1. Molecular signatures observed in the 16S rRNA,
sodA
, and
rpoB
genes in the
S. pneumoniae
complex and in the
S. agalactiae
-
S. difficile
group.
V
OL. 42, 2004
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GENE SEQUENCE-BASED IDENTIFICATION OF COCCI
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[image:5.603.107.482.65.427.2]mined in
S. equinus
, and a 3,651-bp sequence was determined
in
A. defectiva
. After incorporation of these three complete
sequences with those available in GenBank for
S. agalactiae
,
S.
pyogenes
(two sequences in GenBank),
S. pneumoniae
, and
S.
mutans
in the SVARAP software, four variable regions,
char-acterized by lengths of 450 to 750 bp and by a mean variabilitiy
of
⬎
5%, flanked by conserved regions (mean variability,
⬍
5%), were identified in streptococcus
rpoB
genes (numbered
on the basis of the
S. pyogenes rpoB
gene sequence, GenBank
accession number AE006480): region I extended from
posi-tions 1050 to 1800 measured 750-bp in length and exhibited a
2.2 to 8.4% variability; region II extended from positions 1800
to 2250, measured 450 bp, and exhibited a 2.5 to 12.7%
vari-ability; region III extended from positions 2250 to 2750,
mea-sured 500 bp, and exhibited a 2.5 to 13.6% variability; and
region IV extended from positions 2750 to 3500 (750-bp
length; 2.7 to 8.8%) (Table 4). We selected region IV as a
suitable target for identification of clinical isolates. Consensus
PCR primers Strepto 3F and Strepto 3R were designed for
amplification of the region, and an
rpoB
partial sequence
da-tabase analysis was done for an additional 30
Streptococcus
and
related genus species under investigation.
Determination of molecular signatures in
Streptococcus
and
related genera species.
As for the
S. pneumoniae
,
S. mitis
, and
S. oralis
group, we found 15 signatures in
S. pneumoniae
at
positions 115, 121, 157, 193, 285, 313, 320, 331, 355, 517, 520,
559, 562, 577, and 640 of the partial
rpoB
sequence (Fig. 1).
These signatures were all specific for
S. pneumoniae.
As for the
16S rRNA gene, nine bases were unique to
S. pneumoniae
at
positions 29, 172, 218, 238, 592, 711, 820, 984, and 1097 of the
gene sequence. Eight bases were unique to
S. pneumoniae
in
the
sodA
gene at positions 24, 76, 78, 219, 246, 285, 330, and
333 of the gene sequence. PCR incorporating primers
rpoB-pneumoF and rpoBpneumoR yielded an expected 154-bp band
in 10 of 10
S. pneumoniae
isolates. No amplicon was observed
in five of five
S. mitis
isolates and nonspecific amplicons
were obtained in two of five
S.
oralis isolates. As for the
differentiation of
S. agalactiae
from
S. difficile
, three
posi-tions distinguished these two species in partial
rpoB
se-quence: position 139 is an adenine in
S. agalactiae
and a
guanine in
S. difficile
, position 438 is a cytosine in
S.
agalac-tiae
and a thymidine in
S. difficile
, and position 660 is a
guanine in
S. agalactiae
and an adenine in
S. difficile
. A total
of seven positions distinguished these two species in the 16S
rRNA gene sequence.
Results of blind identification testing.
A 740-bp amplicon
was obtained in all of the 102
Streptococcus
sp. isolates
previ-ously identified at the species level and belonging to different
species of
Streptococcus
,
Enterococcus
,
Abiotrophia
,
Gemella
,
and
Granulicatella
genera. Sequence analysis assigned every
one of these isolates to the correct species. Use of the
primer pair Strepto F-Strepto R yielded no amplification
with the isolates belonging to species other than
strepto-cocci with the exception of two
B. cereus
isolates, which
produced an amplicon of the expected size. Sequencing this
amplicon yielded a sequence exhibiting complete identity
with that of
B. cereus rpoB
in GenBank (accession number
AF205342).
DISCUSSION
The data we present here show that PCR amplification of a
740-bp
rpoB
gene fragment by using the primer pair Strepto
F-Strepto R, followed by sequence analysis, is a suitable
mo-lecular approach for the identification of
Streptococcus
,
Entero-coccus
,
Gemella
,
Abiotrophia
, and
Granulicatella
isolates at the
species level. Moreover, this primer pair was shown to be
almost specific for this group of microorganisms, since no
am-plification products were obtained from 58 other bacterial
iso-lates belonging to nonstreptococcal species, including other
gram-positive cocci and species responsible for infectious
en-docarditis. One exception was
B. cereus
, which was amplified
but unambiguously identified by its sequence (39). To date, the
primary method for the molecular identification of
Streptococ-cus
and related genera species has been the analysis of
genomic DNA restriction fragment length patterns on
South-ern blots probed with labeled rRNA genes (10, 18). PCR and
sequencing of rRNA genes, however, have been found to have
limited discriminating power for these species, since the 16S
rRNA gene sequence similarity has been shown to be
⬎
99%
for
S. pneumoniae
,
S. mitis
, and
S. oralis
(22). Also, some
isolates phenotypically and genetically most closely related to
S. mitis
were found to harbor genes encoding the virulence
determinants pneumolysin and autolysin classically associated
with
S. pneumoniae
(51). In contrast, the sequence similarity of
the
groESL
genes was 91.6 to 95.1% in this group of species
(49) and 92 to 96% for
sodA
gene (23, 35). Partial sequence
analysis of the
sodA
gene was shown to discriminate
S.
pneu-moniae
among the mitis group of streptococci (23). As for the
partial
rpoB
gene sequences determined in our study, the
sim-ilarity was in the same order of magnitude of 94%. The
rpoB
gene, then, clearly has a reasonable discriminative power for
this group of
Streptococcus
species. Moreover, we determined
15 signature positions in the 740-bp sequence, which
discrim-inated each one of these three species and served as a basis for
the design of species-specific molecular probes. Partial
rpoB
sequence-based diagnosis with the primers we developed here
could be a suitable alternative for the molecular diagnosis of
streptococcal endocarditis. Morever, the
rpoB
-based system we
developed was able to accurately identify
Enterococcus
,
Ge-mella
,
Abiotrophia
, and
Granulicatella
, four genera comprising
well-known and emerging species also responsible for infective
endocarditis (7, 52). In
Enterococcus
species also, 16S rRNA
gene sequence did not reliabily distinguish isolates at the
spe-cies level, and
groESL
gene-derived PCR primers developed
for
Streptococcus
species did not amplify
Enterococcus
species
(49).
The primer pairs developed here for the detection and
iden-tification of streptococci complement those previously
de-signed for
rpoB
gene-based diagnosis of
Staphylococcus
species
(12). Altogether, 80% of infective endocarditis cases can be
diagnosed by partial
rpoB
gene amplification and sequencing.
Likewise,
S. agalactiae
and
S. difficile
are indistinguishable by
their phenotypic characters, including the protein profile (50),
and share 97.7% similarity in their 16S-23S rRNA spacer, a
high value for streptococci (6).
S. difficile
was not included in a
previous study of partial
sodA
gene sequence for the
identifi-cation of
Streptococcus
species (35). Although these organisms
share an almost identical partial
rpoB
gene sequence, they do
on May 15, 2020 by guest
http://jcm.asm.org/
exhibit three distinct mutations in the
rpoB
sequence that
could serve as a molecular signature for their accurate
identi-fication. In addition to
S. pneumoniae
(8),
S. agalactiae
is a
leading cause of bacterial meningitis, particularly in neonates,
a condition requiring rapid and accurate etiological diagnosis.
With respect to this goal,
rpoB
exhibited
⬎
15% sequence
di-vergence, a value leading to unambiguous species
identifica-tion.
Apart from the 16S rRNA gene-based methods, molecular
tools developed thus far for the identification of
Streptococcus
and related genera did not target these five genera at once as
we did in the present study. Indeed,
groESL
gene-based
tech-niques were restricted to the genus
Streptococcus
(49) and two
different systems based on sequence of the
sodA
gene have
been developped for the genera
Streptococcus
(35) and
Entero-coccus
(36). Likewise, PCR-restriction fragment length
poly-morphism analysis of the entire 16S-23S rRNA region (45)
included 178 strains belonging to 30 species and subspecies of
the genus
Streptococcus
and further studies based on the
anal-ysis of the 16S-23S rRNA intergenic spacer included some
species belonging to the same
Streptococcus
group, such as the
S. mitis
group (2),
S. agalactiae
group (6, 15), and the
S.
ther-mophilus
group (32). The
rpoB
gene-based primer pair
deter-mined in the present study may be helpful for the accurate
detection and identification of
Streptococcus
species and
re-lated genera of medical interest.
ACKNOWLEDGMENT
We acknowledge expert review of the manuscript by Patrick Kelly.
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