JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2008, p. 1019–1025
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 46, No. 3
Genetic Classification and Distinguishing of Staphylococcus Species Based
on Different Partial gap, 16S rRNA, hsp60, rpoB, sodA, and
tuf Gene Sequences?
B. Ghebremedhin,*† F. Layer,† W. Ko ¨nig, and B. Ko ¨nig
Otto-von-Guericke University, Clinical Microbiology, Magdeburg, Germany
Received 23 October 2007/Returned for modification 7 December 2007/Accepted 24 December 2007
The analysis of 16S rRNA gene sequences has been the technique generally used to study the evolution and
taxonomy of staphylococci. However, the results of this method do not correspond to the results of polyphasic
taxonomy, and the related species cannot always be distinguished from each other. Thus, new phylogenetic
markers for Staphylococcus spp. are needed. We partially sequenced the gap gene (?931 bp), which encodes the
glyceraldehyde-3-phosphate dehydrogenase, for 27 Staphylococcus species. The partial sequences had 24.3 to
96% interspecies homology and were useful in the identification of staphylococcal species (F. Layer, B.
Ghebremedhin, W. Ko ¨nig, and B. Ko ¨nig, J. Microbiol. Methods 70:542–549, 2007). The DNA sequence simi-
larities of the partial staphylococcal gap sequences were found to be lower than those of 16S rRNA (?97%),
rpoB (?86%), hsp60 (?82%), and sodA (?78%). Phylogenetically derived trees revealed four statistically
supported groups: S. hyicus/S. intermedius, S. sciuri, S. haemolyticus/S. simulans, and S. aureus/epidermidis. The
branching of S. auricularis, S. cohnii subsp. cohnii, and the heterogeneous S. saprophyticus group, comprising S.
saprophyticus subsp. saprophyticus and S. equorum subsp. equorum, was not reliable. Thus, the phylogenetic
analysis based on the gap gene sequences revealed similarities between the dendrograms based on other gene
sequences (e.g., the S. hyicus/S. intermedius and S. sciuri groups) as well as differences, e.g., the grouping of S.
arlettae and S. kloosii in the gap-based tree. From our results, we propose the partial sequencing of the gap gene
as an alternative molecular tool for the taxonomical analysis of Staphylococcus species and for decreasing the
possibility of misidentification.
The genus Staphylococcus comprises 42 validly described
species and subspecies of gram-positive, catalase-positive cocci
(1, 21, 30), 10 of which contain subdivisions with subspecies
designations (6, 10, 27, 30). Staphylococci, including S. aureus,
generally are opportunistic pathogens or commensals on host
skin. However, they may act as pathogens if they gain entry
into the host tissue through a trauma to the cutaneous barrier,
inoculation by needles, the implantation of medical devices,
or in cases in which the microbial community is disturbed or in
immunocompromised individuals (17–19). Thus, the accurate
species identification of S. aureus as well as that of the other
staphylococcal species in microbial communities is highly de-
sirable to permit a more precise determination of the host-
pathogen relationships of staphylococci (13, 15). The precise
identification of these bacteria to the species level is quite
laborious. Various molecular DNA-based methods for the
identification of Staphylococcus species have been developed.
These methods typically require the use of several species-
specific PCR primers, hybridization probes, or multiple restric-
tion enzymes and usually are not designed to differentiate all
known species simultaneously. 16S rRNA gene sequencing and
PCR-restriction fragment length polymorphism (PCR-RFLP)
analysis have been described for Staphylococcus species iden-
tification (2–4), but these methods do not differentiate between
Staphylococcus lentus and Staphylococcus sciuri. PCR-RFLP
analysis of the 23S rRNA gene with two restriction enzymes is
able to discriminate between Staphylococcus species (23), but
the interpretation of the results is complicated by intervening
sequences (9). More recently, amplified fragment length poly-
morphism fingerprinting has proven to be useful for Staphylo-
coccus species identification, but the method is time-consuming
and expensive (34). Whole-genome DNA-DNA hybridization
analysis (31) allows species identification, but the method is not
suitable for routine use.
The use of nucleic acid targets, with their high sensitivity and
specificity, provides an alternative technique for the accurate
identification and classification of Staphylococcus species. Be-
sides the 16S rRNA gene (2–4), the 16S-23S rRNA intergenic
spacer region (23), and the heat shock protein 60 (hsp60) gene
(11, 12), other gene sequences have been used in genetic stud-
ies: the femA gene (35), the sodA gene (28), the tuf gene (24),
the rpoB gene (5, 26), and the gap gene (36, 37).
In our study, we assessed the usefulness of the ?931-bp
partial sequence of gap for the studied staphylococci (n ? 27)
in species differentiation and for interfering interspecies phy-
logenetic relationships. These are among the most commonly
occurring species of greater clinical significance and are pref-
erentially novobiocin-sensitive staphylococci: e.g., S. aureus, S.
epidermidis, S. warneri, S. haemolyticus, and S. lugdunensis. The
other 15 species that were not subjects of this study are rarely
associated with infections in humans; e.g., S. pasteuri, S. vitu-
linus, and S. saccharolyticus. The gap gene encodes a 42-kDa
transferrin-binding protein (Tpn) located within the cell wall of
* Corresponding author. Mailing address: Otto-von-Guericke University,
Clinical Microbiology, Leipziger Str. 44, Magdeburg, Germany. Phone: 49-
391-6713328. Fax: 49-391-6717802. E-mail: beniam.ghebremedhin@med
† These authors contributed equally to this work.
?Published ahead of print on 3 January 2008.
the staphylococci. Tpn is a member of the newly emerging
family of multifunctional cell wall-associated glyceraldehyde-
3-phosphate dehydrogenases, which is well known for its gly-
colytic function of converting D-glyceraldeyde-3-phosphate to
1,3-bisphosphoglycerate. gap commonly has been considered a
constitutive housekeeping gene (8, 25).
Yugueros and coworkers published the sequences of the gap
genes of 12 staphylococcal species relevant for humans (36).
We extended these studies and sequenced the ?931-bp se-
quence encoding a partial region of the gap gene from a total
of 27 different staphylococcal species (22). We consider these
species to be among the clinically significant species, as do
other groups (2–5, 12, 28).
MATERIALS AND METHODS
Bacterial strains and growth conditions. All of the staphylococcal strains were
grown on blood agar and incubated at 37°C for 18 to 24 h. Reference strains were
selected from the German Collection of Microorganisms and Cell Cultures
(DSMZ), the Czech Collection of Microorganisms (CCM), and the American
Type Culture Collection (ATCC), and they included the following: Staphylococ-
cus arlettae DSM 20672T, S. aureus ATCC 29213T, S. carnosus subsp. carnosus
DSM 20501T, S. cohnii subsp. cohnii DSM 20260T, S. delphini DSM 20771T, S.
epidermidis DSM 20044T(CCM 2124T), S. equorum subsp. equorum DSM
20674T, S. hyicus DSM 20459T, S. intermedius DSM 20373T(CCM 5739T), S.
kloosii DSM20676T, S. lugdunensis DSM 4804T(ATCC 43809T), S. warneri DSM
20316 (CCM 2730T), S. capitis subsp. capitis CCM 2734T, S. caprae CCM 3573T,
S. chromogenes CCM 3387T, S. gallinarum CCM 3572T, S. haemolyticus CCM
1798T, S. hominis subsp. hominis CCM 2732T, S. lentus CCM 3472T, S. muscae
CCM 4175T, S. saprophyticus subsp. saprophyticus CCM 883T, S. sciuri CCM
3473T, S. simulans CCM 2705T, S. xylosus CCM 2725T, S. auricularis ATCC
33753T, S. felis ATCC 49168T, and S. schleiferi subsp. schleiferi ATCC 43808T.
Isolation of genomic DNA. Chromosomal DNA was isolated from overnight
cultures grown on blood agar at 37°C. Genomic DNA was extracted by using the
Qiagen DNA extraction kit according to the manufacturer’s suggestions (Hilden,
Germany), with the modification that 20 ?l of lysostaphin (1 mg/ml; Sigma) and
20 ?l lysozyme (100 mg/ml; Qiagen) were added at the cell lysis step. The
concentration of the DNA was assessed spectrophotometrically.
DNA sequencing. Consensus gap PCR primers (Table 1) were used as previ-
ously described (22). Gap1-for and Gap2-rev were used to amplify the ?931-bp
fragment as described before (37), and the PCR products were purified with the
Qiagen gel extraction kit (Hilden, Germany). Partial reverse and forward se-
quencing of the ?931-bp fragment was obtained by using the consensus primers
at 3.25 pmol (Table 1). Sequencing reactions were carried out with the BigDye
Terminator v3.1 cycle sequencing kit (Applied Biosystems) according to the
manufacturer’s instructions and using the previously described sequencing pro-
tocols (22). The results were processed into sequence data with sequence analysis
software (Applied Biosystems), and partial sequences were combined into a
single consensus sequence with assembler software (Applied Biosystems) (22).
The gene sequences other than that of gap were obtained from GenBank (Ta-
Phylogenetic analysis. Sequences were aligned manually in Sequencher 3.0
(Gene Codes Corporation) to edit the sequences, if necessary, and to note which
regions were to be excluded for the phylogenetic analysis. Multiple-sequence
alignments and topology predictions were done with DNASISMAX, version
2.0.5 (2003) (Hitachi Software Engineering, Japan). Phylogenetic trees were
generated with the neighbor-joining algorithm by using DNASISMAX. All trees
were resampled with 1,000 bootstrap replications to ensure the robustness of the
data (7). The phylogenetic analyses were displayed with the TreeView drawtree
program, version 1.6.6 (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).
The DNA sequence similarity analysis was performed with BioEdit, version
PCR, sequencing of the gap gene, and sequence similarity
for the staphylococcal species. The amplification of the partial
gap gene for all of the Staphylococcus species yielded a single
product of nearly 931 bp. The GenBank accession numbers are
DQ321674 to DQ321700.
The sequence similarity of the gap sequences ranged from
24.3 to 96% (Table 3). The species S. lentus and S. sciuri
revealed a sequence similarity of 93% according to the gap
gene sequences, whereas the other gene-based similarities
ranged between 88 and 98% (88% for hsp60, 88.9% for sodA,
and 98% for 16S rRNA). The species S. capitis and S. caprae
revealed a sequence similarity of 27% according to the gap
gene sequences, whereas the other gene-based similarities
ranged between 94 and 99% (94% for sodA, 95% for 16S
rRNA, and 99% for hsp60). The species S. carnosus and S.
simulans revealed a sequence similarity of 95% according to
the gap gene sequences, whereas the other gene-based similar-
ities ranged between 86 and 98% (87 to 88% for rpoB and 96%
for 16S rRNA).
The species S. chromogenes and S. hyicus revealed a se-
quence similarity of 91% according to the gap gene sequences,
whereas the other gene-based similarities ranged between 77.6
and 98% (77.6% for sodA and 98% for 16S rRNA).
Staphylococcus phylogeny derived from gap sequences. The
Staphylococcus species were divided into three clades (Fig. 1)
with significant bootstrap values (?90%); the first contained
the S. hyicus/S. intermedius group, comprising S. hyicus, S.
chromogenes, S. delphini, and S. intermedius. The second clade
contained two groups, the S. sciuri group, comprising S. sciuri
and S. lentus, and the S. haemolyticus/S. simulans group, com-
prising S. haemolyticus, S. xylosus, S. muscae, S. simulans, S.
schleiferi subsp. schleiferi, S. carnosus subsp. carnosus, S. caprae,
and S. felis. The third clade contained only one group, the S.
aureus/S. epidermidis group, comprising S. aureus, S. hominis
subsp. hominis, S. warneri, S. epidermidis, S. capitis subsp.
capitis, and S. lugdunensis. The branching of S. auricularis, S.
cohnii, and the heterogeneous S. saprophyticus group, compris-
ing S. saprophyticus subsp. saprophyticus, S. equorum subsp.
equorum, S. gallinarum, S. arlettae, and S. kloosii, was not reli-
able (bootstrap values of 75, 75, and 29%, respectively).
For the gap gene comparison between S. sciuri and S. lentus,
a sequence similarity value of 82% (Table 3) was determined,
as the position of these species in the phylogenetic tree is
supported by a bootstrap value of 100% (see below).
TABLE 1. Primers used for sequencing the gap gene
aPosition relative to the S. aureus gap sequence.
bPrimer sequences from Yugueros et al. (36).
cDesigned primer sequences from this study.
1020 GHEBREMEDHIN ET AL.J. CLIN. MICROBIOL.
Comparative phylogeny based on various gene sequence-
derived trees. The gap-derived sequence similarity analysis for
the staphylococcal species is given in Table 3. According to the
staphylococcal gene sequences of gap, rpoB, and sodA, the
species S. felis and S. muscae grouped within the same cluster,
whereas in the 16S rRNA- and hsp60-derived trees these two
species did not show a close relationship.
The species S. hyicus and S. chromogenes clustered together
according to the 16S rRNA, hsp60, and rpoB sequence analysis,
but the clustering by sodA analysis was less close. The grouping
of S. hyicus and S. chromogenes was more affirmed by the gap
gene than by the other genes as well.
The grouping of S. arlettae and S. kloosii in the gap-based
tree (with bootstrap values of 98%) was fairly supported by
hsp60-, rpoB-, sodA-, and 16S rRNA-derived trees (bootstrap
values of ?43%).
S. hominis subsp. hominis and S. lugdunensis clustered into
the same group according to gap (bootstrap value of 95%), tuf
(bootstrap value of 44%), and hsp60 (bootstrap value of 29%)
analyses, whereas this was not observed for 16S rRNA, rpoB,
and sodA analyses.
The grouping of S. delphini and S. intermedius was supported
by gap, 16S rRNA, hsp60, and sodA analyses. The phylogenetic
relationship of S. chromogenes and S. hyicus in the gap- and
rpoB-derived trees (for each bootstrap value of 100%) was
supported by low bootstrap values in hsp60-, 16S rRNA-, and
sodA-derived trees. The close relationship of S. carnosus subsp.
carnosus and S. simulans in gap (bootstrap value of 100%),
hsp60, sodA (for each bootstrap value of 98%), and rpoB (boot-
strap value of 79%) analyses was confirmed as well. S. delphini
and S. intermedius clustered together according to gap, 16S
rRNA, sodA, and hsp60 analyses, with bootstrap values of
Several molecular targets have been exploited for the mo-
lecular identification of Staphylococcus species. Because a
large amount of rrs sequence data is available in a public
database, it is not surprising that the 16S rRNA gene has been
an obvious choice. Gene sequence-based identification of bac-
teria at the species level may require resolving the whole gene,
yet in some cases, phylogenetically closely related bacterial
species cannot be differentiated from each other. Although the
comparison of the 16S rRNA gene sequences has been useful
in phylogenetic studies at the genus level, its use has been
questioned in studies at the species level. In this regard, the
16S rRNA sequence similarity has been shown to be very high,
90 to 99%, in 29 Staphylococcus species (20). S. caprae and S.
capitis cannot be distinguished by their 16S rRNA gene se-
quences (34). Similarly, some Staphylococcus taxa have the
same 16S rRNA gene sequences in variable regions V1, V3,
V7, and V9, with identical sequences occurring in, e.g., S.
vitulinus, S. saccharolyticus, S. capitis subsp. urealyticus, S.
caprae, the two subspecies of S. aureus, and the two subspecies
of S. cohnii (34). Other gene sequences have been used em-
pirically in attempts to classify the Staphylococcus species,
namely, the hsp60 gene, the sodA gene, the rpoB gene, and the
tuf gene. With regard to the hsp60 gene, it should be noted that
the cloned partial hsp60 gene DNA sequences of nine isolates
TABLE 2. Partial gene sequences from GenBank used for phylogenetic tree
GenBank sequence for:
16S rRNA hsp60 rpoB sodA tuf
S. capitis subsp. capitis
S. carnosus subsp. carnosus
S. cohnii subsp. cohnii
S. equorum subsp. equorum
S. hominis subsp. hominis
S. saprophyticus subsp.
S. schleiferi subsp. schleiferi
VOL. 46, 2008 SEQUENCE-BASED IDENTIFICATION OF STAPHYLOCOCCUS SPP. 1021
of S. aureus showed a mean variability of only 2% (33). Also,
cross-hybridization occurred in cloned partial hsp60 genes be-
tween the DNAs from S. intermedius and S. delphini (12). The
gap sequences were less conserved compared to the above-
mentioned genes (sequence similarities, 24 to 96%). There-
fore, the gap gene is rather more discriminative, as shown for
S. caprae and S. capitis, which were clearly distinguished from
each other, in contrast to results from 16S rRNA gene analy-
ses. For the sodA gene, a pairwise comparison of these se-
quences revealed a mean sequence similarity of 81.5%, which
was lower than that calculated for the rrs sequences of staph-
ylococci (98%). The rpoB, hsp60, and tuf partial sequences
showed interspecific similarity values of 71.6 to 93.6, 74 to 93,
and 86 to 97%, respectively.
The gap-based tree indicates the divergence of the selected
staphylococcal species, which was well supported for most of
the strains studied. We compared the gap-derived tree (Fig. 1)
to those inferred from sequences of the 16S rRNA, rpoB, sodA,
hsp60, and tuf genes of Staphylococcus species available from
GenBank (Table 2). Earlier studies on the taxonomy of Staph-
ylococcus species based on DNA-DNA reassociation indicated
that in this genus there were eight distinct species groups,
represented by S. epidermidis, S. saprophyticus, S. simulans, S.
intermedius, S. hyicus, S. sciuri, S. auricularis, and S. aureus (17,
18). The same groups were identified in a study using hsp60
(20) and the sodA gene sequence analysis (28). The phyloge-
netic tree generated from rpoB sequences revealed nine clus-
ters, including an additional S. haemolyticus group. The 16S
rRNA sequence-derived trees with 38 taxa of the genus Staph-
ylococcus identified 11 genogroups (S. epidermidis, S. sapro-
phyticus, S. simulans, S. carnosus, S. hyicus/S. intermedius, S.
sciuri, S. auricularis, S. warneri, S. haemolyticus, S. lugdunensis,
and S. aureus) (32, 33). However, the bootstrap values for most
of the nodes of the distinct clusters were low. With the gap
sequences and a bootstrap value of ?90%, the Staphylococcus
species were divided into four well-supported clusters: the S.
sciuri group, the S. hyicus/S. intermedius group, the S. haemo-
lyticus/S. simulans group, and the S. aureus/S. epidermidis
In hsp60-, 16S rRNA-, sodA-, and rpoB-generated trees, the
S. sciuri group was formed by the two members S. sciuri and S.
lentus with bootstrap values of ?97%. All of the species from
the S. sciuri group, including S. vitulinus (not included in this
study), differ from the other Staphylococcus species in several
remarkable features. They are novobiocin resistant and oxi-
dase positive, and they all share the same characteristic pattern
of amino acid substitution in their hsp60 proteins (12, 20). The
close relationship between S. sciuri and S. lentus was repro-
duced in the results from our tree analysis based on gap se-
quences. Thus, members of the S. sciuri group form a constant
cluster in hsp60, 16S rRNA, sodA, rpoB, and gap gene trees.
The S. hyicus/intermedius group, as defined by 16S rRNA
sequence analysis and confirmed by rpoB and hsp60 sequence
analysis, includes S. hyicus, S. chromogenes, S. muscae, S. in-
termedius, S. delphini, S. schleiferi subsp. schleiferi, and S. felis.
The S. intermedius group, as defined by sodA sequence analysis,
consisted of S. intermedius and S. delphini (bootstrap value of
100%). S. schleiferi subsp. schleiferi and S. felis were not in-
cluded in this cluster. Moreover, the related species S. hyicus,
S. muscae, and S. chromogenes did not cluster to form an S.
TABLE 3. DNA sequence identity matrix based on comparisons of the gap gene sequences of the Staphylococcus species
Taxon (gap gene no.)
% Identity with gap gene no.a:
123456789 101112 1314 1516 17 181920 212223 2425 2627
S. arlettae (1)
S. aureus (2)
S. auricularis (3)
S. capitis (4)
S. caprae (5)
S. carnosus (6)
S. chromogenes (7)
S. cohnii (8)
S. delphini (9)
S. epidermidis (10)
S. equorum (11)
S. felis (12)
S. gallinarum (13)
S. haemolyticus (14)
S. hominis (15)
S. hyicus (16)
S. intermedius (17)
S. kloosii (18)
S. lentus (19)
S. lugdunensis (20)
S. muscae (21)
S. saprophyticus (22)
S. schleiferi (23)
S. sciuri (24)
S. simulans (25)
S. warneri (26)
S. xylosus (27)
1022GHEBREMEDHIN ET AL.J. CLIN. MICROBIOL.
FIG. 1. Neighbor-joining tree based on the 931- to 933-bp gap sequences and 16S rRNA, rpoB, sodA, and tuf gene sequences showing the
phylogenetic relationships among the staphylococcal species selected for this study. The value on each branch is the percent occurrence of the
branching order in bootstrapped trees (7).
VOL. 46, 2008 SEQUENCE-BASED IDENTIFICATION OF STAPHYLOCOCCUS SPP.1023
hyicus species subgroup. The gap-derived data support the idea
that S. chromogenes and the non-S. aureus coagulase-positive
staphylococci, such as S. intermedius, S. delphini, and S. hyicus,
belong to the S. hyicus/S. intermedius species group. In our
study, S. schleiferi subsp. schleiferi and S. muscae were outside
of the S. hyicus/S. intermedius group as well. Based on the
gap-derived data, S. schleiferi subsp. schleiferi and S. muscae
could be grouped into the S. haemolyticus/S. simulans group.
According to the 16S rRNA gene sequence, the phylogenetic
classification of S. felis fell into the clade of S. hyicus/S. inter-
medius. However, based on DNA-DNA reassociation studies,
S. felis was thought to be related to members of the S. simulans
group (17, 18). In our gap-derived data, S. felis is outside of the
S. hyicus/S. intermedius group and is closely related to members
of the S. simulans group.
In contrast to the 16S rRNA sequence-based phylogenetic
tree but in accordance with the sodA, hsp60, and rpoB gene
sequences, we did not divide S. simulans and S. carnosus into
two closely related groups by using the gap gene-derived se-
The S. saprophyticus group, as defined by 16S rRNA sequence
analysis, includes the novobiocin-resistant and oxidase-negative
species S. saprophyticus subsp. saprophyticus, S. arlettae, S. kloosii,
The rpoB-derived data indicated that S. cohnii is outside of the S.
saprophyticus group. From the sodA-derived data, one could con-
clude that the monophyly of this clade is uncertain, since it is
associated with a bootstrap value of only 68%. In our study, S.
cohnii and S. xylosus are clearly outside of the S. saprophyticus
group. S. cohnii belongs to the S. saprophyticus group according to
the 16S rRNA and hsp60 trees. Based on gap-derived sequences,
the branching of S. auricularis, S. cohnii, and the heterogeneous S.
saprophyticus group, comprising S. saprophyticus subsp. sapro-
phyticus, S. equorum subsp. equorum, S. gallinarum, S. arlettae, and
S. kloosii, was not reliable (98, 70, 43, and 36%, respectively).
Based on the 16S rRNA data, the S. epidermidis species
group was divided into five cluster groups, as described by
Kloos (16): S. lugdunensis, S. haemolyticus, S. warneri, S. epi-
dermidis, and S. aureus (33). The S. epidermidis cluster, com-
posed of S. epidermidis, S. capitis, S. caprae, and S. saccharo-
lyticus (not included in our study), constitutes a monophyletic
clade supported by a high bootstrap value (97%) on the basis
of 16S rRNA sequence analysis (20). In the rpoB study, S.
caprae and S. capitis appeared to be in the S. haemolyticus
group. Similarly to the S. saprophyticus group, the S. epidermi-
dis group did not form a clearly distinct lineage in the sodA-
based study (bootstrap value of 38.9%). Similar results were
obtained in our study using gap-based sequences. Moreover, in
our study, S. caprae showed no close relationship to S. epider-
midis or S. capitis. On the other hand, the association of S.
warneri with the S. epidermidis group was inferred from our
data as well as from the rpoB tree analysis.
Obviously, the determination of the sequences of several
genes is an important tool for pathogen identification and
phylogenetic studies. Although each gene-derived tree will dif-
fer from the others and will have different levels of statistical
support, it has been found that groupings obtained with two
different sequences with bootstrap values of ?90% are stable
and reliable (29). The data we present show that the sequence
analysis of a 931-bp gap gene fragment is a suitable molecular
method for the identification of Staphylococcus isolates at the
The gap gene sequence-based relationships of the staphylo-
coccal species obtained were in accordance with phylogenetic
trees published previously (37). In support of the gap-derived
tree results, the tuf gene sequence-derived tree indicates that S.
warneri is associated with the S. epidermidis group, which in-
cludes S. capitis. This is in agreement with results described
earlier for sequencing assays targeting the sodA gene (28). The
tuf gene-derived data often showed more intraspecies se-
quence divergence than the 16S rRNA-derived data. Appar-
ently, the 16S rRNA gene is more highly conserved than the tuf
gene. A pairwise comparison of the tuf gene sequences re-
vealed that their mean identity (92.6%) is lower than the mean
identity (95.9%) of 16S rRNA gene sequences. These results
indicate that the tuf gene constitutes a more discriminatory
target gene than the 16S rRNA gene to differentiate closely
related Staphylococcus species.
The phylogenetic analysis of the staphylococcal gap se-
quences yields an evolutionary tree having a topology similar
to that of the tree constructed with the 16S rRNA sequences,
although minor differences were observed (Fig. 1).
We have determined the gap sequences of 27 Staphylococcus
type strains and demonstrated the usefulness of the gap
GenBank database for distinguishing the staphylococcal spe-
cies and giving an approach for interpreting the phylogenetic
relationship of the staphylococci. This method consists of a
PCR carried out with a single pair of degenerate oligonucleo-
tides for the amplification of a staphylococcal partial gap gene
sequence and the direct sequencing of the amplicon. The se-
quencing also can be performed with the two respective PCR
primers instead of the eight primers, as was done in this study,
since the sequencer is adjusted for sequence analysis. In our
case, the sequencer also was used for terminal RFLP analysis,
so that we had troubles with the sequencing of fragments of
?800 bp to obtain a confident sequence properly. The meth-
odology might be useful in reference laboratories for the char-
acterization of strains that could not be assigned to a species on
the basis of their conventional phenotypic reaction and can
stand on its own more effectively than 16S rRNA analysis, as
this is a highly conserved gene and has limited discriminatory
power compared to that of the gap gene, especially in closely
related staphylococcal species. Shortening the region of inter-
est within the gap gene sequence was not possible due to the
scattering of the divergent regions throughout the whole se-
quence. However, gap sequencing did not allow the detection
of intraspecies polymorphism among the studied Staphylococ-
cus species; e.g., for Staphylococcus epidermidis, the sequence
similarity among different isolates of this species was more
than 99%. This also was shown by the gap-based terminal
RFLP analysis of S. epidermidis isolates in our previous publi-
This study was supported by the German Federal Ministry of Edu-
cation and Research (BMBF NBL3).
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