Phylogenetic Analyses of Based on the 16S rDNA Sequence and Assignment of Clinical Isolates from Animals

Tatsufumi TAKAHASHI, Masayoshi KANEKO, Yukari MORI, Masayoshi TSUJI1), Naoya KIKUCHI, and Takashi HIRAMUNE Departments of Epizootiology and 1)Experimental Animals, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu 069, Japan

(Received 18 February 1997/Accepted 12 May 1997)

ABSTRACT. The nucleotide sequences of the 16S rDNA in 17 strains of 16 taxa of the genus Staphylococcus were determined. The sequences were compared phylogenetically together with the gene sequences of 10 (including 7 other species) Staphylococcus species retrieved from the DNA Data Bank of Japan. Although the primary and secondary structures of most of Staphylococcus species were very similar (homology values 96.4% or more) except for S. caseolyticus MAFF 911387T (homology values 95.4% or less), the 23 staphylococcal species were divided into 10 groups based on similarity, evolutionary distance and phylogenetic tree analysis. Nucleotide stretches in several variable domains in the 16S rDNA sequence appeared to be specific for the bacterial groups or the species. By comparing such characteristics in the sequence and phylogenies of 5 staphylococcal clinical isolates from bovine mastitis, canine and feline pyoderma, and feline urogenital syndrome with the information obtained in this study, the species level of each organism was identified. — KEY WORDS: rDNA, 16S ribosomal RNA, Staphylococcus. J. Vet. Med. Sci. 59(9): 775–783, 1997

Thirty-six taxa (species and subspecies) of the genus have indicated the existence of species-specific nucleotide Staphylococcus are listed in the American Type Culture stretches within the gene [3, 28, 39], however, the number Collection (ATCC) catalogue, updated in June 1996. Four of the species determined their sequence were limited. species and three subspecies of Staphylococcus have been The aim of this study was to detect subtle differences added to the list of as newly-identified organisms between staphylococcal species by comparing their sequence from studies conducted in the 1990s [5, 14, 22, 26, 37, 38, patterns of the 16S rDNA and to understand the phylogenetic 40]. Some of these studies employed sequence analyses of relationships. We also examined the usefulness of the ribosomal RNA gene (rDNA) or its transcript (rRNA) comparative gene analysis for precise identification of [14, 40]. The importance of genetic relationships among staphylococcal isolates in the veterinary field. bacterial species has been established, and rDNA analysis is the most effective method available for determining MATERIALS AND METHODS phylogenetic relationships as well as for identification of microorganisms, due to its reproducibility [29]. Particularly, Bacterial strains: The bacterial strains that were used for the integration of small-subunit rDNA sequence data has the 16S rDNA analysis are shown in Tables 1 and 2. Table made possible the assignment of any bacterial organism by 1 is a list of Staphylococcus strains obtained from the the standard technique, nucleotide sequencing of small DNA American Type Culture Collection (ATCC) or from the fragments. On the other hand, using a phenotypic Ministry of Agriculture, Forestry and Fisheries, Japan identification system such as the API STAPH system or a (MAFF). The 16S rDNA sequences of staphylococcal combination of several recommended biological tests [23, strains and Micrococcus agilis DSM 20550 retrieved from 25], the precise assignment of a staphylococcal strain to the the DNA Data Bank of Japan (DDBJ) are also listed. species level may be difficult or even impossible if the strain Although the sequence of S. aureus subsp. aureus ATCC has atypical characteristics. Due to the limited number of 12600T has already been reported by Ludwig et al. [27], we stable discriminating characteristics, many of the taxa determined the sequence of the same strain to confirm the remain difficult to distinguish from one another by reliability of the direct sequencing method employed in this phenotypic tests. Precise identification of not only study. The scientific names for the strains of Staphylococcus pathogens of veterinary importance such as S. aureus, S. were taken from the ATCC catalogue, updated in October intermedius, and S. hyicus, which are known as - 1996. Table 2 shows five staphylococcal strains that were positive bacteria, but also coagulase-negative staphylococcal isolated and maintained in our laboratory. These strains, species (CNS) has been laborious, costly, and not always which were identified as S. aureus (S. intermedius) and S. accurate [9, 10, 13], although there has been increasing simulans by the API STAPH identification system (Bio recognition of the ability of CNS to produce clinically Mérieux S. A., Marcy-I’Etoile, France), were used for a significant infections [23, 25, 32]. Earlier studies on the detailed comparative analysis of the 16S rDNA sequences. of species of the genus Staphylococcus, using Each of these strains was grown on a trypticase soy agar 16S (small subunit for bacteria) rDNA sequence analyses, plate incorporating 5% bovine blood under an atmosphere 776 T. TAKAHASHI, ET AL.

Table 1. Staphylococci used for the sequence analysis of the 16S rDNA

Bacteria Accession no. of 16S rDNA Species groupa) Species Strain sequence

S. aureus S. aureus subsp. aureus ATCC 12600Tc) D83357 S. aureus subsp. aureusb) ATCC 12600T X68417 S. aureus subsp. aureusb) NCDO 949 X70648 S. aureus subsp. anaerobius ATCC 35844T D83355 S. epidermidis S. epidermidis ATCC 14990T D83363 S. epidermidis MAFF 911486 (ATCC 146) D83362 S. capitis subsp. capitisb) ATCC 27840T L37599 S. saccharolyticusb) ATCC 14953T L37602 S. warnerib) ATCC 27836T L37603 S. haemolyticus MAFF 911476 (ATCC 29970)T D83367 S. haemolyticusb) CCM 2737 X66100 S. hominisb) DSM 20328 X66101 S. saprophyticus S. cohnii subsp. cohnii MAFF 911487 (ATCC 29974)T D83361 S. saprophyticus MAFF 911473 (ATCC 15305)T D83371 S. saprophyticusb) NT 75 L20250 S. xylosus MAFF 911482 (ATCC 29971)T D83374 S. gallinarum ATCC 35539T D83366 S. hyicus S. hyicus ATCC 11249T D83368 S. chromogenes MAFF 911474 (ATCC 43764)T D83360 S. intermedius S. intermedius MAFF 911388 (ATCC 29663)T D83369 S. schleiferi subsp. schleiferib) ATCC 43808T S83568 S. simulans S. simulans MAFF 910161 (ATCC 27848)T D83373 S. felis ATCC 49168T D83364 S. sciuri S. sciurib) ATCC 29062T S83569 S. lentus MAFF 911385 (ATCC 29070)T D83370 S. caseolyticus S. caseolyticus MAFF 911387 (ATCC 13548)T D83359 Undetermined S. auricularis MAFF 911484 (ATCC 33753)T D83358 S. muscaeb) CCM 4175 (ATCC 49910)T S83566 M. agilisb) DSM 20550 X80748

a) Species groups were designated according to the references [24, 25]. b) Nucleotide sequence retrieved from the DDBJ. c) Type strain of the taxon (species or subspecies).

Table 2. Staphylococcal strains used for the identification based on the 16S rDNA sequences

Interpretation of Accession no. Strain Origin the results with of 16S rDNA API STAPH system sequence

Kitami Cattle, mastitis S. aureus D83354 OA1 Cattle, mastitis S. aureus D83356 CD22-1 Dog, pyoderma S. aureus (S. intermedius)a) D83372 FD21-2 Cat, pyoderma S. simulans D83365 FU16A2 Cat, feline urological S. simulans D83353 syndrome

a) Possibility to S. intermedius by forming pigmentless colonies and isolation from veterinary sample.

of 5% CO2 at 37˚C for 24 hr. chain reaction (PCR). To amplify the 16S rDNA, PCR was Sequence determination of 16S rDNAs: The 16S rDNA performed with a pair of generic primers for Gram-positive sequences of Staphylococcus strains were determined. The bacteria, primer A(+) (5’ AGAGTTTGATCCTGGCTC 3’) genomic DNA was extracted from a single colony grown and primer B (5’ ggttaccttgttacgactt 3’), according to basic on a plate culture using a DNA extraction kit (SMITEST, protocols [33]. During the PCR amplification, biotin was Sumitomo Kinzoku Kogyo Co., Ltd., Tokyo, Japan) introduced into one of the strands of the PCR product using according to the manufacturer’s instructions. The 16S rDNA one of the primers biotinylated in the 5’ end. The PCR was amplified from the extracted DNA by the polymerase products of the DNA were then converted into a single- PHYLOGENY OF STAPHYLOCOCCUS 16S rDNA 777 stranded template by immobilization onto ferrous beads distance of 0.001) than to the type strain of S. epidermidis (Dynabeads, Dynal A. S., Oslo, Norway) with coupled (99.45% and 0.005, respectively). streptavidin on the surface and denaturation with NaOH, as S. caseolyticus was the most distant, with 95.03% described by Hultman et al. [18, 19]. The immobilized similarity and an evolutionary distance of 0.052, from S. template was used for the Sanger dideoxy DNA sequencing aureus subsp. aureus ATCC 12600T (type strain of the type modified by Zimmermann et al. [41], with a panel of species of the genus). This organism was dissimilar to oligonucleotide primers designed for Gram-positive bacteria. other strains with a range of similarity of 93.94–95.44% The 5’ termini of the sequence primers were labeled with and evolutionary distance values of 0.047–0.065. The next fluorescein isothiocyanate, and the procedures for distant species, S. simulans, S. sciuri, and S. lentus, exhibited polyacrylamide gel electrophoresis and determining DNA approximately 97% similarity and approximately 0.03 sequences were performed according to the manufacturer’s evolutionary distance from other species except for S. instructions of the A.L.F. DNA Sequencer II (Pharmacia caseolyticus. Biotech, Upsala, Sweden). Some sequences seemed to be grouped with higher Sequence alignment, calculation of similarity values, and similarities. For example, S. epidermidis, S. saccharolyticus phylogenetic tree construction: The nucleotide sequences of and S. capitis subsp. capitis, whose percent similarities were the 16S rDNA determined in this study were aligned 99.39% or more within the group, showed similarities of manually along with the sequences retrieved from the DDBJ 98.78% or less with other sequences. Such sequence groups (Table 1). Evolutionary distance (Knuc) values were were notable in S. haemolyticus and S. hominis, and in S. determined by using the Clustal V program package [16]. sciuri and S. lentus, although grouping for other sequences Percent similarity between individual sequences (A and B) based on the similarity values were not clear. was calculated as follows: similarity (A, B) = 100 × sum of Phylogenies of staphylococcal 16S rDNAs: The sequence the matches/(length – gap residues (A) – gap residues(B)). groups, which are more closely related to each other than to The neighbor-joining method of Saitou and Nei [34] was the other sequences based on similarity values, were also employed to construct a phylogenetic tree using the apparent in the phylogenetic tree. Two subspecies of S. BioResearch SINCA program package (Fujitsu, Japan). The aureus were specifically related to each other (bootstrap topology of the tree was evaluated by a bootstrapping value 100%). S. sciuri and S. lentus also exhibited a specific method [12]. association (bootstrap value 100%) as did a cluster of S. Nucleotide sequence accession number: The nucleotide epidermidis, S. sacchalolyticus and S. capitis subsp. capitis sequence data reported in this paper appear in the GSDB, (bootstrap value 98%). S. saprophyticus, S. xylosus, S. DDBJ, EMBL and NCBI nucleotide sequence databases gallinarum and S. cohnii subsp. cohnii formed a with the accession numbers listed in Tables 1 and 2. monophyletic group that was recovered in 93% of the bootstrapped tree. Two bacterial groups, one formed by S. RESULTS muscae, S. felis, S. chromogenes, S. hyicus, S. intermedius and S. schleiferi subsp. schleiferi, and the other formed by Nucleotide sequences of staphylococcal 16S rDNAs: In S. hominis and S. haemolyticus, exhibited relatively close this study, nucleotide sequences of the 16S rDNA were genealogical affinity (bootstrap values of 90 and 80%, determined for 16 type strains of the named taxa of genus respectively). Each of the other staphylococcal species, S. Staphylococcus, one non-type strain of S. epidermidis, and caseolyticus, S. simulans, S. auricuralis or S. warneri, 5 field isolates. The sequences were aligned with each appeared to form a relatively deep subline within the genus other and compared with the staphylococcal 16S rDNA Staphylococcus. We designated these groups of species as sequences available in previously published papers [3, 14, “cluster groups” of aureus, epidermidis, warneri, 27, 40] or in the DDBJ. The alignment was done from haemolyticus, saprophyticus, hyicus/intermedius, auricuralis, nucleotide residues 26 to 1,491, based on the Escherichia sciuri, simulans and caseolyticus (Fig. 1 and Table 3). The coli numbering system [4]. The 1,476-bases length of the criteria for typing of the cluster group are as follows; 1) sequence of S. aureus subsp. aureus ATCC 12600T Bootstrap value is 80% or more at the node of the cluster, (D83357) employed for the gene analysis corresponds to 2) Neither 90 or more percent similarity nor 0.01 or less 1,487 bases of the length of multiple alignment (including evolutionary distance between two cluster groups, 3) A large gaps). clade including strains of S. aureus and S. epidermidis was Similarity values of staphylococcal 16S rDNAs: Table 3 subgrouped into two cluster groups due to the medical/ shows percent similarities and evolutionary distance values veterinary importance of S. aureus. for combinations of the 16S rDNA sequences in different Phylogenetic positions of staphylococcal isolates in the species of the genus Staphylococcus. In the table, where veterinary field: Five staphylococcal strains isolated from the sequences were arranged by the order of the similarities, animals with suppurative diseases were used for comparative the similarities were quite high among sequences of the analysis of the 16S rDNA (Table 2). To confirm the results same species except for S. epidermidis. Strain MAFF of the genetical assignments, these isolates were used for 911486 of the species was more similar to S. capitis subsp. examinations of biochemical properties using the API capitis ATCC 27840T (99.93% similarity and evolutionary STAPH system. Furthermore, biophysiological tests were 778 T. TAKAHASHI, ET AL.

Fig. 1. Unrooted phylogenetic tree based on 16S rDNA sequences for the species of the genus Staphylococcus and Micrococcus agilis as an out-group. The numbers are the estimated confidence levels, expressed as percentages, for the positions of the branches, determined by bootstrap analysis [12]. Bootstrapping values less than 70% are not shown. Scale bar indicates the evolutionary distance value (Knuc) between sequences, determined by measuring the lengths of the horizontal lines connecting two organisms. “Cluster group” was designated based on the branching pattern and percent similarity (Table 3). done for the isolates and for the type strains of S. chromogenes strains [21]. The positive result of the Staphylococcus species. The examinations were performed coagulase test and negative reaction with in strain for acidification activities from , maltose, , FU16A2 were also inconsistent with the interpretation of and using the PYP medium supplemented the API STAPH. The 16S rDNA sequences of the feline with each of the hydrocarbonate, activity, resistence isolates were clearly different from each other; the strain to and , and for coagulase test. FD21-2 was nearly identical to the type strain of S. felis, Strains Kitami and OA1, isolated from mastitis milk of while the strain FU16A2 was located within the cluster of individual cases were identified as S. aureus by the API S. aureus strains. These subgroups were recovered in 100% STAPH system. These isolates were novobiocin sensitive, of the bootstrapped tree. bacitracin resistent and positive for the coagulase test. These Species or group specific sequence in the 16S rDNA of formed typical yellowish round colonies on a Staphylococcus: The 16S rDNA nucleotide stretches staphylococcus-medium no.110 agar plate. These results possibly specific for a certain species or a cluster group of are typical for S. aureus subsp. aureus. While isolate CD22- the genus Staphylococcus were summarised in Table 4. The 1 from a case of canine pyoderma, whose profile in the API table shows 5 regions of the sequences, which seems to be STAPH system was interpreted as S. aureus (or S. useful for the assignment of staphylococcal strains. The intermedius if the isolate was from a veterinary specimen), strains of S. aureus used were identical each other at all of grew as distinct pigmentless colonies and reacted negatively these regions, and two regions, residues 72–97 and 183– with maltose and trehalose. Some of these propeties were 203 were specific for the cluster group. The strains in the inconsistent with those of S. aureus or S. intermedius. In cluster group of epidermidis shared same nucleotide the phylogenetic tree (Fig. 1), the 16S rDNA sequences of stretches in these regions except for two changes found at bovine isolates were located within the clusters of S. aureus, residue 201 in S. epidermidis ATCC 14990T and at 1,005 in while the canine isolate formed a distinct subgroup with S. S. capitis subsp. capitis ATCC 27840T whose change was schleiferi subsp. schleiferi (bootstrap value 95%). found only in each of the strains. The cluster group specific Two feline isolates, FD21-2 and FU16A2 from the sequences were found in the heamolyticus and the sciuri, diseased parts of the pyoderma and cystitis, respectively, while partially specific stretches were found between the were interpreted as S. simulans by the API STAPH system. cluster groups of epidermidis and simulans at residues 72– However, the isolate FD21-2 exhibited sensitivity to 97. In the cluster groups of saprophyticus and intermedius/ bacitracin, which is observed limitedly in S. felis, S. capitis, hyicus, group specific stretch was not detected, however, S. warneri, S. haemolyticus, S. hominis, S. caseolyticus and there were specific sequences for one or several species PHYLOGENY OF STAPHYLOCOCCUS 16S rDNA 779

,

T

S.

S.

AFF

, 14.

M

T

ATCC 11249

028 0.062

031 0.057

037 0.057

.030 0.059

.032 0.064

0.029 0.060

0.037 0.047 0.035 0.048

MAFF 911486, 7.

S. caseolyticus

.36 94.83 J

.58 I 0.054

MAFF 911473

S. hyicus

0.008

, 28.

T

, 20.

T

99.18

S. epidermidis

, 6.

S. saprophyticus

T

MAFF 910161

MAFF 911388

ATCC 14990

S. simulans

0.014 0.017 0.023 0.031 0.032 0.035 0.060

DSM 20328*, 13.

, 27.

S. intermedius

T

0.012 0.014 0.017 0.020 0.033 0.036 0.036 0.063

, 19.

T

S. epidermidis

S. hominis

0.012 0.012 0.014 0.016 0.024 0.033 0.036 0.036 0.063

, 5.

T

MAFF 911385

ATCC 43808

0.008

99.11 0.010

S. lentus

CCM 2737*, 12.

ATCC 35844

99.19 0.010

schleiferi

, 26.

T

0.011 0.033 0.030 0.029 0.031 0.033 0.028 0.025 0.025 0.033 0.031 0.060

0.011 0.033 0.029 0.028 0.030 0.033 0.030 0.026 0.028 0.033 0.033 0.057

0.011 0.030 0.025 0.024 0.024 0.028 0.027 0.023 0.029 0.031 0.031 0.061

subsp.

anerobius

S. haemolyticus

0.012 0.013 0.033 0.031 0.030 0.031 0.034 0.030 0.027 0.028 0.034 0.033 0.060

ATCC 29062

subsp.

, 11.

T

S. schleifieri

saprophyticus; F. hyicus/intermedius; G, auricularis; H, sciuri, I, simulans; J, caseodyticus. b) *Sequence data

99.18

S. sciuri

, 18.

S. aureus

T

0 or less.

14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

, 25.

0.002 0.003 0.010

98.85

T

MAFF 911476

0.011 0.013 0.013 0.028 0.013 0.028 0.027 0.025 0.024 0.029 0.021 0.022 0.027 0.032 0.025 0.059

99.80 0.005 99.73 99.52 0.008

99.05

MAFF 911487

NCDO 949*, 4.

MAFF 911484

0.012 0.015 0.017 0.017 0.027 0.017 0.031 0.031 0.029 0.027 0.033 0.024 0.026 0.031 0.036 0.029 0.063

cohnii

aureus

S. haemolyticus

0.004 0.009

98.90 0.017 0.019 0.019 0.019 0.019 0.028 0.029 0.026 0.023 0.029 0.021 0.023 0.029 0.031 0.028 0.061

subsp.

, 10.

T

subsp.

S. auricularis

99.59 99.12

S. cohnii

S. aureus

0.012 0.014 0.019 0.014 0.023 0.025 0.024 0.024 0.022 0.027 0.029 0.026 0.024 0.026 0.019 0.024 0.027 0.028 0.026 0.060

0.015 0.018 0.022 0.018 0.024 0.026 0.025 0.026 0.024 0.026 0.027 0.024 0.026 0.027 0.024 0.022 0.029 0.031 0.028 0.061

0.016 0.020 0.024 0.020 0.026 0.028 0.027 0.016 0.026 0.026 0.029 0.026 0.028 0.029 0.026 0.024 0.031 0.033 0.030 0.065

0.017 0.019 0.023 0.019 0.024 0.026 0.025 0.020 0.024 0.026 0.028 0.024 0.026 0.028 0.024 0.023 0.030 0.030 0.028 0.062

, 17.

ATCC 27836

T

*, 3.

T

CCM 4175T*, 24.

Swarneri

, 9.

ATCC 35539

T

ATCC 12600

S. muscae

0.005 0.006 0.006

, 23.

aureus

T

S. gallinarum

ATCC 27840

0.014 0.014 0.016 0.014 0.017 0.016 0.020 0.021 0.023 0.025 0.021 0.026 0.024 0.031 0.033 0.029 0.028 0.030 0.027 0.027 0.026 0.030 0.030 0.052

0.014 0.014 0.016 0.014 0.017 0.016 0.020 0.021 0.023 0.025 0.021 0.026 0.024 0.030 0.032 0.028 0.027 0.029 0.026 0.027 0.026 0.030 0.030 0.052 0.014 0.014 0.017 0.015 0.017 0.015 0.019 0.020 0.022 0.024 0.021 0.026 0.023 0.030 0.032 0.028 0.027 0.031 0.026 0.026 0.026 0.029 0.029 0.052 0.014 0.013 0.017 0.014 0.018 0.017 0.021 0.021 0.024 0.026 0.022 0.027 0.024 0.031 0.034 0.034 0.029 0.031 0.028 0.028 0.027 0.031 0.031 0.052

99.45 0.005 0.001

99.46 99.53 0.005

99.39 99.93 99.46

subsp.

, 16.

T

capitis

ATCC 49168

S. aureus

subsp.

, 2.

0.002 0.007

S. felis

T

MAFF 911482

, 22.

T

S. capitis

0.000 0.002 0.007

c)

, 8.

T

a)

12345678910111213

S. xylosus

ATCC 12600

A

99.80 99.79 0.003 99.93 99.93 99.73 98.65 98.61 98.58 98.58 B

98.64 98.67 98.57 98.64

98.31 98.31 98.30 98.30

98.50 98.50 98.49 98.49

98.23 98.23 98.22 98.22 98.78 98.37 98.23 98.37 C 0.015 0.019 0.016 0.018 0.020 0.017 0.021 0.018 0.028 0.024 0.023 0.024 0.028 0.022 0.017 0.027 0.029

95.03 94.98 94.97 94.97 94.21 94.34 93.98 94.02 94.23 94.35 94.00 94.14 94.29 94.28 94.47 94.15 94.01 94.01 94.08 94.19 94.49 94.53 94.24 93.94 95.44 95

98.44 98.40 98.51 98.38 98.31 98.57 98.10 98.16 98.37 D

98.04 97.98 98.18 97.97 97.83 98.22 97.49 97.62 97.89 97.97 97.91 98.12 97.91 98.24 98.57 97.97 98.03 98.10

97.77 97.70 97.84 97.70 97.70 97.75 97.29 97.55 98.30 98.82 98.51 98.31 E 97.55 97.49 97.70 97.49 97.48 97.54 97.10 97.28 98.10 98.71 98.36 98.16 97.88 97.83 97.95 97.81 97.68 97.68 97.22 97.48 98.30 98.70 98.29 98.08

97.44 97.35 97.51 96.70 97.44 97.67 97.16 97.21 98.23 98.44 98.04 97.98

97.66 97.60 97.73 97.59 97.66 97.79 97.36 97.55 98.16 98.69 98.27 98.76 98.90 98.69 98.90 98.90 0.030 0.024 0.026 0.026 0.031 0.028 0.019 0.029 0.033 0.

97.08 97.01 97.18 97.01 97.49 97.34 97.43 97.48 97.21 98.70 98.29 98.08 96.74 96.92 96.79 97.08 97.11 F

96.97 96.80 96.50 96.90 97.37 97.20 97.22 97.28 97.62 97.36 96.96 97.23 97.09 97.01 97.13 97.59 97.59

97.20 97.19 97.26 97.13 97.61 97.47 97.49 97.69 97.76 97.54 97.13 97.40 97.13 97.13 97.20 97.58 97.59 98.77

97.31 97.24 97.31 97.24 97.45 97.59 97.29 97.48 97.62 97.66 97.31 97.72 97.11 96.96 97.04 97.59 97.45 98.76 98.76 98.97 0.014 0.017 0.023 0.033 0.034 0.

97.13 97.05 96.99 97.06 97.33 97.47 97.15 97.28 97.21 97.13 96.78 97.12 96.72 96.65 96.79 97.20 96.90 98.56 98.56 98.56 98.54 0.019 0.025 0.032 0.035 0. 97.30 97.31 97.47 97.24 97.65 98.06 97.70 97.75 97.76 97.93 97.66 98.00 97.24 97.20 97.03 97.37 97.17 98.47 98.27 98.27 98.47 98.13 0.026 0.031 0.033 0

97.37 97.35 97.44 97.30 97.84 97.68 97.63 97.82 98.23 97.83 97.50 97.77 97.56 97.35 97.47 97.77 98.07 97.62 98.04 97.74 97.73 97.56 97.37 G 0.031 0.034 0

97.49 97.35 97.58 97.42 97.15 97.34 96.89 96.94 97.21 97.35 97.11 97.24 97.15 97.38 97.27 97.15 97.11 96.92 96.68 96.99 96.76 96.85 97.00 97.01 H

97.06 97.06 97.13 96.99 96.99 97.27 96.55 96.74 96.95 96.86 96.45 96.92 96.79 96.72 96.79 96.93 96.77 96.45 96.45 96.92 96.76 96.58 96.68 96.72 97.10 96.94 97.17 97.03 97.37 97.48 97.02 97.14 97.07 97.56 97.16 97.29 96.96 96.74 96.79 96.97 97.25 96.54 96.56 96.58 96.97 97.02 97.03 96.89 96.40 96

100.00

*

*

*

MAFF 911474

aureus

*

*

ATCC 14953

*

b)

*

NT 75*, 15.

*

*

*

*

subsp.

, a) Framed data indicate the values within a certain cluster group; A. aureus; B. epidermidis; C. warneri; D, haemolyticus; E.

T

S. caseolyticus

S. haemolyticus

S. haemolyticus

S. hominis S. saprophyticus

S. xylosus

S. saprophyticus

S. gallinarum

S. cohnii

S. schleiferi

S. intermedius

S. hyicus

S. chromogenes

S. felis

S. muscae S. auricularis

S. sciuri

S. lentus S. simulans

S. aureus

S. aureus

S. aureus

S. epidermidis

S. saccharolyticus

S. capitis

S. warneri

S. epidermidis

S. anaerobius

S. chromogenes

S. aureus

1.

2.

3. 4. 5. 6.

7.

8.

9.

28.

10.

11.

12. 13.

15.

14.

16.

17.

18.

19.

20.

21.

22.

23. 24.

25.

26. 27.

Table 3. Percent similarity (lower left) and evolutionary distance (upper right) for the 16S rDNA sequences of staphylococci

saccharolyticus

21. saprophyticus

1. 911387 were retrieved from the DDBJ. c) Bold-typed data indicate percent similarity of 99.00 or more, or evolutionary distance of 0.01 780 T. TAKAHASHI, ET AL.

Table 4. Staphylococcal 16S rDNA nucleotide stretches specific for species or cluster group

Staphylococcus strain 72–97a) 183–203 999–1022 1117–20 1290–93

S. aureus subsp. aureus ATCC 12600T GGACGAGAA-GCTTGC-TTCTCTGAT b) TATTTTGAACCGCATGGTTCAAAAG CTTTGACAACTCTAGAGATAGAGCC AAGC GTTG S. aureus subsp. aureus NCDO 949 GGACGAGAA-GCTTGC-TTCTCTGAT TATTTTGAACCGCATGGTTCAAAAG CTTTGACAACTCTAGAGATAGAGCC AAGC GTTG S. aureus subsp. anaerobius ATCC 35844T GGACGAGAA-GCTTGC-TTCTCTGAT TATTTTGAACCGCATGGTTCAAAAG CTTTGACAACTCTAGAGATAGAGCC AAGC GTTG

S. epidermidis ATCC 14990T AGACGAGGA-GCTTGC-TCCTCTGAC CATGTTGAACCGCATGGTTCAATAG CTCTGACCCCTCTAGAGATAGAGTT AAGC GTTG S. epidermidis MAFF 911487 AGACGAGGA-GCTTGC-TCCTCTGAC CATGTTGAACCGCATGGTTCAACAG CTCTGACCCCTCTAGAGATAGAGTT AAGC GTTG S. capitis subsp. capitis ATCC 27840T AGACGAGGA-GCTTGC-TCCTCTGAC CATGTTGAACCGCATGGTTCAACAG CTCTGATCCCTCTAGAGATAGAGTT AAGC GTTG S. saccharolyticus ATCC 14953T AGACGAGGA-GCTTGC-TCCTCTGAC CATGTTGAACCGCATGGTTCAACAG CTCTGACCCCTCTAGAGATAGAGTT AAGC GTTG

S. warneri ATCC 27836T AGATAAGGA-GCTTGC-TCCTTTGAC CATATTGAACCGCATGGTTCAATAG CTTTGACCGCTCTAGAGATAGAGTC AAGC GTTG

S. hominis DSM 20328 AGACGAGGA-GCTTGC-TCCTTTGACc) TATTTCGAACCGCATGGTTCGATAG CTTTGACCCTTCTAGAGATAGAAGT AAGC GTTG S. haemolyticus MAFF 911476T AGACAAGGA-GCTTGC-TCCTTTGAC TATTTCGAACCGCATGGTTCGATAG CTTTGACAACTCTAGAGATAGAGCC AAGC GTTG S. haemolyticus CCM 2737 AGACAAGGA-GCTTGC-TCCTTTGAC TATTTCGAACCGCATGGTTCGATAG CTTTGACAACTCTAGAGATAGAGCC AAGC GTTG

S. cohnii subsp. cohnii MAFF 911487T AGATAAGGA-GCTTGC-TCCTTTGAC CATTTAGAACCGCATGGTTCTAAAG CTTTGACAACTCTAGAGATAGAGCC AAAC GTTG S. gallinarum ATCC 35539T AGATAAGGA-GCTTGC-TCCTTTGAC CATATAGAACCGCATGGTTCTATAG CTTTGACCACTCTAGAGATAGAGCT AAGC GTTG S. xylosus MAFF 911482T AGATAAGGA-GCTTGC-TCCTTTGAA CATTTAGAACCGCATGGTTCTAAAG CTTTGAAAACTCTAGAGATAGAGCC AAGC GTTG S. saprophyticus MAFF 911473T AGATAAGGA-GCTTGC-TCCTTTGAC CATTTGGAACCGCATGGTTCTAAAG CTTTGAAAACTCTAGAGATAGAGCC AAGC GTTG S. saprophyticus NT 75 AGATAAGGA-GCTTGC-TCCTTTGAC CATTTGGAACCGCATGGTTCTAAAG CTTTGAAAACTCTAGAGATAGAGCC AAGC GTTG

S. muscae CCM 4175T AGACGAGGT-GCTTGC-ACCTCTGAC TATATTGAACCGCATGGTTCAATAG CTTTGACCGCACTAGAGATAGTGTT GAGC GTTG S. felis ATCC 49168T TGAAGAGGA-GCTTGC-TCCTTTGAC TATGTTGAACCGCATGGTTCAACAG CTTTGACCGCTCTAGAGATAGAGTT AAGC GTTG S. chromogenes MAFF 911474T TGACGAGGA-GCTGGC-TCCTTTGAC CATATCGAACCGCATGGTTCGATAG TTTTGACCACTCTAGAGATAGAGTT GAGC GCCG S. hyicus ATCC 11249T AGATGAGGA-GCTTGC-TCCTTTGAC CATGTTGAACCGCATGGTTCACTAG TTTTGATCGCTCTAGAGATAGAGTT AAGC GTTG S. intermedius MAFF 911388T AGATAAGGA-GCTTGC-TCCTTTGAC CATGTTGAACCGCATGGTTCTACAG CTTTGACCGCTCTAGAGATAGAGTT GAAC GTTG S. schleiferi subsp. schleiferi ATCC 43808T GGACGAGGA-GCTTGC-TCCTTTGAA CATGTTGAACCGCATGGTTCAACAG CTTTGACCGCTCTAGAGATAGAGTT GAGC GTTG

S. auricularis MAFF 911484T AGATAAGGA-GCTTGC-TCCTTTGAC CATGTTGAACCGCATGGTTCTACAG CTTTGACAACTCTAGAGATAGAGTC AAGC GTTG

S. lentus MAFF 911385T AGATGAGAA-GCTTGC-TTCTCTGAT TATATTGAACCGCATGGTTCAATGT CTTTGATCGCTCTAGAGATAGAGTT AAGC ATTA S. sciuri ATCC 29062T AGATGAGAA-GCTTGC-TTCTCTGAT TATTTTGAACCGCATGGTTCAATAG CTTTGACCGCTCTAGAGATAGAGTC AAGC ATTA

S. simulans MAFF 910161T AGACGAGGA-GCTTGC-TCCTCTGAC CACATGAAACCGCATGGTTTCATGA CTTTGACAACTCTAGAGATAGAGCT AAGC GTTG

S. caseolyticus MAFF 911387T GGACGAGAGTGCTTGCACTCTCTGAT TATTTAGCTTCGCATGAAGCAATAG CTTTGACAACTCTAGAGATAGAGCT ATCT ACCA a) Nucleotide residues according to the Escherichia coli numbering system [4], b) Framed sequence(s) was found specifically in certain cluster group(s), c) Bold-typed sequence was found specifically in certain strain or species.

included in the cluster group. Contrary, specific stretch for intermedius plus S. hyicus, respectively. The species group a certain species was not found in S. epidermidis, S. was defined not only by the entire genomic DNA association saccharolyticus, S. intermedius, or two subspecies of S. but also by extensive phenotypic character analysis [25]. aureus at the domains covered in Table 4. Of the biological properties, novobiocin resistance clearly divided the cluster groups of saprophyticus and sciuri from DISCUSSION others. Oxydase-positive species, S. sciuri, S. lentus, and S. caseolyticus were also distinct from other species in our The relatively close relationship among species of the phylogenetic tree. However, the other well-described genus Staphylococcus was confirmed by phylogenetic phenotypic characteristics, coagulase and β-galactosidase analyses based on the gene sequencing employed in this activities, were not shared by all species within a specific study. Another genetic approach in bacterial taxonomy is cluster group, hyicus/intermedius or saprophyticus. These nucleic acid pairing analysis of an entire genome such as results confirm that novobiocin resistance and oxydase DNA-DNA hybridization and the thermal stability of DNA activity are essential phenotypes for the classification of heteroduplexes. Based on the integration of chromosomal biotypes such as species groups within the genus DNA pairing analysis [24, 25, 35], a dendrogram of the Staphylococcus. In other words, there is only a limited DNA relationship was drawn and six species groups and number of stable discriminating characteristics to distinguish several independent species are currently recognized, one taxon supported phylogenetically from another by represented by the species of S. epidermidis, S. phenotypic tests [17]. saprophyticus, S. simulans, S. intermedius, S. hyicus, S. Although our cluster groups correlated well with the sciuri, S. aureus, and S. caseolyticus [24, 25]. Most of the species groups and other genealogical taxonomies such as branching patterns forming the cluster groups agreed with ribotypes [6–8], several disagreements were found. Two the above-mentioned dendrogram. The cluster groups of established species, S. felis and S. simulans, which are epidermidis, warneri and haemolyticus corresponded with indistinguishable by several species differentiation tables of the species group of S. epidermidis, as did the cluster groups phenotypic tests [17, 20, 23–25] except for bacitracin of saprophyticus, sciuri and intermedius/hyicus with the resistance [21], were distinct in the phylogenetic tree with a species groups of S. saprophyticus, S. sciuri, and S. relatively deep branch. In contrast, two subspecies of the PHYLOGENY OF STAPHYLOCOCCUS 16S rDNA 781 species of S. aureus, aureus and anaerobius, were coagulans, has not been determined yet, we could not assign phenotypically different to each other [17], and this was the strain into the subspecies level. The positive result of supported by the results from ribotyping [7], however, their this strain in the coagulase test and negative results in 16S rDNA sequences were nearly indistinguishable in this maltose and trehalose acidification indicated that the study. A nucleotide change was found at only one residue organism is subsp. coagulans [22]. Strain FD21-2, isolated between type strains of the subspecies. This disagreement from a specimen of subcutaneous abscess of a cat, was may be due to the ribotyping probe having non-coding clearly identified as S. felis with one nucleotide change from region(s) adjacent to the 16S rRNA gene [7]. Dolzani et al. the type strain of the species. Both the strain were bacitracin [11] showed that the 16S-23S rRNA intergenic spacer sensitive and the identical result in the API STAPH system regions of S. aureus strains were highly variable in length. except for mannitol acidification. Although the system On the other hand, restriction analysis using pulsed-field doesn’t support the profiles for S. felis strains, biochemical gel electrophoresis showed that genomic relatedness characteristics of these strains agrees to those described by between the type strains of subspecies aureus and Igimi et al. [21]. This organism has been isolated from cats anaerobius was ca. 20%, while the most distant strains diagnosed as suffering from a variety of skin infections such within the subspecies aureus was ca. 30% [30]. as wound abscesses or external ear otitis [20, 21]. Although Stackebrandt and Goebel [36] reported that organisms that the etiological role of this organism is still not clear, the have less than 97.0% homology of rRNA will not reassociate assignment based on the specific nucleotide stretches in the to more than 60% of genomic relatedness, no matter which 16S rDNA could be helpful for understanding the ecology hybridization method is applied. This is not the case vice of S. felis. versa, however, several combinations of strains of either We encountered another confusion in the phylogenies of mycobacteria or Fibrobacter with 99% or more homology 16S rDNAs among the strains in the cluster group of of rRNA sequence showed less than 50% relatedness of epidermidis (Table 3, Table 4 and Fig. 1). Although there DNA reassociation [1, 2]. These findings indicate that is a possibility that the strain MAFF 911486 of S. highly homologous 16S rDNAs do not always reflect epidermidis is actually a strain of S. capitis, paying more genealogically close relatedness. However, branching attentions for the identification of the strains in these taxa is patterns in a phylogenetic tree based on the 16S rDNA necessary. The extensive analysis of the organisms in their sequence were more reliable than by the other genetic typing phenotypical and genealogical characteristics are also analysis, if the bootstrap value was sufficiently high. In the required. present study, the degree of nucleotide changes among In this study, we determined the 16S rDNA sequence for species of the genus Staphylococcus was relatively small, about half of the species of the genus Staphylococcus. but the bootstrapping tree of the species was recovered in However, species specificity of the sequence is still not high probabilities. This suggests that the sequence diversity clear. Furthermore, for the limited number of the strains of the gene is stable and such a stretch could be species- or used for the analysis, the branching pattern in the taxon-specific. The results of multiple sequence alignment phylogenetic tree may change at the nodes with low of staphylococci showed substantial domains where specific bootstrap values. Therefore, it is necessary to complete the stretches were frequently detected (Table 4). These domains 16S rDNA sequence database for all of the type strains of were found in and around variable regions, particularly in the undetermined taxa of the genus Staphylococcus and to the V1, V3 and V7 regions. The specificities of these determine the sequence for more strains in each taxon. stretches for the species or a certain group of the genus Further searching for such sequences is in progress. Staphylococcus are still not clear since the 16S rDNA Detecting a specific sequence to identify pathogenic agents sequences in 13 species/subspecies of the genus have not is more useful for studies on staphylococcal infections, been determined. However, most of the species exhibited because standard and commonly used biochemical tests do specific stretches in the domains suggesting that these are not correctly identify the number of strains, due to useful for the precise identification of staphylococcai. phenotypic divergence [15, 31]. Sequence analysis of about In the present study, we were able to specify the 1,500 nucleotides has become rapid and inexpensive [36]. phylogenetic positions of 16S rDNA in clinical isolates as This improvement has made the sequence comparison of S. aureus, S. schleiferi and S. felis. Furthermore, each 16S rDNA much easier to perform than DNA-DNA sequence of the isolates were identical to that of each species hybridization or ribotyping. We have specified the at the nucleotide stretches shown in Table 4. However, we phylogenetic positions of five veterinary isolates by could not differenciate the two subspecies of S. aureus, sequence comparison, although due to the limitation of aureus and anaerobius, due to the limitation of sequence information for undetermined taxa, we could not discriminating power by 16S rDNA sequence, while an identify the subspecies level of the isolates. However, isolate from canine superficial pyoderma, CD22-1, was further efforts towards the construction of a sequence nearly indistinguishable from the type strain of S. schleiferi database will make the 16S rDNA sequencing the most subsp. schleiferi in their 16S rDNA sequences (four effective method for bacterial classification. Additional nucleotide changes were detected). Since the 16S rDNA analyses such as the sequencing of 16S–23S intergenic sequence of another subspecies, S. schleiferi subsp. spacer regions, which are more variable than 16S rDNA for 782 T. TAKAHASHI, ET AL. closely related taxa, should then be employed for further 15. Hedin, G. 1994. Comparison of genotypic and phenotypic precise identification [11]. These sequencing techniques methods for species identification of coagulase-negative sta- are extremely helpful in deciding whether the laborious and phylococci isolates from blood cultures. APMIS 102: 855–864. unstable test needs to be performed. 16. Higgins, D. G., Bleasby, A. J., and Fuchs, R. 1992. Clustal V: improved software for multiple sequence alignment. Comput. Appl. Biosci. 8: 189–191. ACKNOWLEDGEMENTS. This work was supported by a 17. Holt, J. G., Krieg, N. R., Sneath, P. H. A., Staley, J. T., and grant-in-aid for Scientific Research (08760281) from the Williams, S. T. (eds.) 1994. Bergey’s Manual of Determina- Ministry of Education, Science and Culture of Japan and by tive Bacteriology, 9th ed., The Williams and Wilkins Co., a grant-in-aid to cooperative research from Rakuno Gakuen Baltimore. University, 1995. 18. Hultman, T., Bergh, S., Moks, T., and Uhlén, M. 1991. Bidi- rectional solid phase sequencing of in vitro-amplified plasmid REFERENCES DNA. Bio Techniques 10: 84–93. 19. Hultman, T., Ståhl, S., Hornes, E., and Uhlén, M. 1989. Di- 1. Amann, R. I., Lin, C., Key, R., Montgomery, L., and Stahl, rect solid phase sequencing of genomic and plasmid DNA D. A. 1992. Diversity among Fibrobacter strains: towards a using magnetic beads as solid support. Nucleic Acids Res. 17: phylogenetic classification. Syst. Appl. Microbiol. 15: 23–32. 4937–4946. 2. Baess, I. 1983. Deoxyribonucleic acid relationships between 20. Igimi, S., Atobe, H., Tohya, Y., Inoue, A., Takahashi, E., and different serovars of Mycobacterium avium, Mycobacterium Konishi, S. 1994. Characterization of the most frequently intracellulare and Mycobacterium scrofulaceum. Acta Pathol. encountered Staphylococcus sp. in cats. Vet. Microbiol. 39: Microbiol. Immunol. Scand. Sect. B 91: 260–296. 255–260. 3. Bentley, R. W., Harland, N. M., Leigh, J. A., and Collins, M. 21. Igimi, S., Kawamura, S., Takahashi, E., and Mitsuoka, T. D. 1993. A Staphylococcus aureus-specific oligonucleotide 1989. Staphylococcus felis, a new species from clinical speci- probe derived from 16S rRNA gene sequences. Lett. Appl. mens from cats. Int. J. Syst. Bacteriol. 39: 373–377. Microbiol. 16: 203–206. 22. Igimi, S., Takahashi, E., and Mitsuoka, T. 1990. Staphylo- 4. Brosius, J., Palmer, J. L., Kennedy, J. P., and Noller, H. F. coccus schleiferi subsp. coagulans subsp. nov., isolated from 1978. Complete nucleotide sequence of a 16S ribosomal RNA the external auditory meatus of dogs with external ear otitis. gene from Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 75: Int. J. Syst. Bacteriol. 40: 409–411. 44801–44805. 23. Kloos, W. E. and Bannerman, T. L. 1995. Staphylococcus 5. Chesneau, O., Morvan, A., Grimont, F., Labischinski, H., and and Micrococcus. pp. 282–298. In: Manual of Clinical Mi- El Solh, N. 1993. Staphylococcus pasteuri sp. nov., isolation crobiology, 6th ed. (Murray, P. R., Baron, E. J., Pfaller, M. from human, animal, and food specimens. Int. J. Syst. A., Tenover, F. C., and Yolken, R. H. eds.), ASM Press, Bacteriol. 43: 237–244. Washington, D. C. 6. Cookson, B. D., Stapleton, P., and Ludlam, H. 1992. 24. Kloos, W. E. and Schleifer, K.-H. 1986. Genus IV Staphylo- Ribotyping of coagulase-negative staphylococci. J. Med. coccus. pp. 1013–1035. In: Bergey’s Manual of Systematic Microbiol. 36: 414–419. Bacteriology, vol. 2 (Sneath, P. H. A., Mair, N. S., Sharpe, 7. de Buyser, M.-L., Morvan, A., Aubert, S., Dilasser, F., and M. E., and Holt, J. G. eds.), The Williams and Wilkins Co., El Solh, N. 1992. Evaluation of a ribosomal RNA gene probe Baltimore. for the identification of species and subspecies within the 25. Kloos, W. E., Schleifer, K.-H., and Götz, R. 1991. The genus genus Staphylococcus. J. Gen. Microbiol. 138: 889–899. Staphylococcus. pp. 1369–1420. In: The Prokaryotes: a Hand- 8. de Buyser, M.-L., Morvan, A., Grimont, F., and El Solh, N. book on the Biology of Bacteria: Ecophysiology, Isolation, 1989. Characterization of Staphylococcus species by riboso- Identification, Applications, 2nd ed. (Balows, A., Trüper, H. mal RNA gene restriction patterns. J. Gen. Microbiol. 135: G., Dworkin, M., Harder, W., and Schleifer, K.-H. eds.), 989–999. Springer-Verlag, New York. 9. Deinhofer, M. and Pernthaner, A. 1995. Staphylococcus spp. 26. Kloos, W. E. and Wolfshohl, J. F. 1991. Staphylococcus cohnii as mastitis-related pathogens in goat milk. Vet. Microbiol. subspecies: Staphylococcus cohnii subsp. cohnii subsp. nov. 43: 161–166. and Staphylococcus cohnii subsp. urealyticum subsp. nov. 10. Devriese, L. A., Laevens, H., and Haesebrouck, F. 1994. A Int. J. Syst. Bacteriol. 41: 284–289. simple identification scheme for coagulase negative staphy- 27. Ludwig, W., Kirchof, G., Klugbauer, N., Weizenegger, M., lococci from bovine mastitis. Res. Vet. Sci. 57: 240–244. Betzl, D., Ehrmann, M., Hertel, C., Jilg, S., Tatzel, R., 11. Dolzani, L., Tonin, E., Lagatolla, C., and Monti-Bragadin, C. Zitzelsberger, H., Lievl, S., Hochberger, M., Shah, J., Lane, 1994. Typing of Staphylococcus aureus by amplification of D., and Wallnoef, P. R. 1992. Complete 23S ribosomal RNA the 16S-23S rRNA intergenic spacer sequence. FEMS sequences of Gram-positive bacteria with a low DNA G+C Microbiol. Lett. 119: 167–174. content. Syst. Appl. Microbiol. 15: 487–501. 12. Felsenstein, J. 1985. Confidence limits on phylogenies: an 28. Ludwig, W., Schleifer, K.-H., Fox, G. E., Seewaldt, E., and approach using bootstrap. Evolution 39: 783–791. Stackebrandt, E. 1981. A phylogenetic analysis of staphylo- 13. Greene, R. T. and Lämmler, Ch. 1993. Staphylococcus inter- cocci, Peptococcus saccharolyticus and Micrococcus medius: current knowledge on a pathogen of veterinary mucilaginosus. J. Gen. Microbiol. 125: 357–366. importance. J. Vet. Med. B 40: 206–214. 29. Olsen, G. J. and Woese, C. R. 1993. Ribosomal RNA: a key 14. Hájek, V., Ludwig, W., Schleifer, K. H., Springer, N., to phylogeny. FASEB J. 7: 113–123. Zitzelsberger, W., Kroppenstedt, R. M., and Kocur, M. 1992. 30. Pantucek, R., Götz, F., Doskar, J., and Rosypal, S. 1996. Staphylococcus muscae, a new species isolated from flies. Genomic variability of Staphylococcus aureus and the other Int. J. Syst. Bacteriol. 42: 97–101. coagulase-positive Staphylococcus species estimated by PHYLOGENY OF STAPHYLOCOCCUS 16S rDNA 783

macrorestriction analysis using pulsed-field gel electrophore- J. Syst. Bacteriol. 44: 846–849. sis. Int. J. Syst. Bacteriol. 46: 216–222. 37. Tanasupawat, S., Hashimoto, Y., Ezaki, T., Kozaki, M., and 31. Pennington, T. H., Harker, C., and Thomson-Carter, F. 1991. Komagata, K. 1992. Staphylococcus piscifermentans sp. nov., Identification of coagulase-negative staphylococci by using from fermented fish in Thailand. Int. J. Syst. Bacteriol. 42: sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 577–581. rRNA restriction patterns. J. Clin. Microbiol. 29: 390–392. 38. Webster, J. A., Bannerman, T. L., Hubner, R. J., Ballard, D. 32. Pfaller, M. A. and Hervaldt, L. A. 1988. Laboratory, clinical, N., Cole, E. M., Bruce, J. L., Fiedler, F., Schubert, K., and and epidemiological aspects of coagulase-negative staphylo- Kloos, W. E. 1994. Identification of the Staphylococcus sciuri cocci. Clin. Microbiol. Rev. 1: 281–299. species group with EcoRI fragments containing rRNA se- 33. Saiki, R. K. 1990. Amplification of genomic DNA. pp. 13– quences and description of Staphylococcus vitulus sp. nov. 20. In: PCR Protocols, a Guide to Methods and Applications Int. J. Syst. Bacteriol. 44: 454–460. (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J. 39. Zakrzewska-Czerwinska, J., Gaszewska-Mastalarz, A., eds.), Academic Press, San Diego. Pulverer, G., and Mordarski, M. 1992. Identification of Sta- 34. Saitou, N. and Nei, M. 1987. The neighbor-joining method: a phylococcus epidermidis using a 16S rRNA-directed new method for reconstructing phylogenetic trees. Mol. Biol. oligonucleotide probe. FEMS Microbiol. Lett. 100: 51–58. Evol. 4: 406–425. 40. Zakrzewska-Czerwinska, J., Gaszewska-Mastalarz, A., Lis, 35. Schleifer, K. H., Meyer, S. A., and Rupprecht, M. 1979. B., Gamian, A., and Nordarski, M. 1995. Staphylococcus Relatedness among coagulase-negative staphylococci: deoxy pulvereri sp. nov., isolated from human and animal speci- ribonucleic acid reassociation and comparative immunologi- mens. Int. J. Syst. Bacteriol. 45: 169–172. cal studies. Arch. Microbiol. 122: 93–101. 41. Zimmermann, J., Voss, H., Schwager, C., Stegemann, J., Erfle, 36. Stackebrandt, E. and Goebel, B. M. 1994. Taxonomic note: a H., Stucky, K., Kristensen, T., and Ansorge, W. 1990. A place for DNA-DNA reassociation and 16S rRNA sequence simplified protocol for fast plasmid DNA sequencing. Nucleic analysis in the present species definition in bacteriology. Int. Acids Res. 18: 1067.