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

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Phylogenetic Analyses of Staphylococcus Based on the 16S Rdna Sequence and Assignment of Clinical Isolates from Animals Phylogenetic Analyses of Staphylococcus 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 bacteria 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 coagulase- 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. taxonomy 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
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