|
|
||||||||
1 Department of Pathology, Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium
2 BCCM/LMG Bacteria Collection, Laboratory of Microbiology, Faculty of Sciences, Ghent University, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
3 Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Ghent University, De Pintelaan 185, B-9000 Gent, Belgium
Correspondence
Marc Vancanneyt
Marc.Vancanneyt{at}UGent.be
| ABSTRACT |
|---|
|
|
|---|
-haemolysin production, the pathogenic significance of the novel species remains unclear. The type strain, LMG 22219T (=ON 86T=CCUG 49543T), was isolated from lung tissue of a cat.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains LMG 22219T, LMG 22220, LMG 22221 and LMG 22222 are AJ780976, AJ780977, AJ780978 and AJ780979, respectively.
| MAIN TEXT |
|---|
|
|
|---|
|
The phylogenetic position of all four strains (LMG 22219T to LMG 22222) was determined by 16S rRNA sequence analysis. DNA was extracted as indicated for tRNA intergenic length polymorphism analysis. To amplify the 16S rRNA gene, a PCR was performed using the conserved primers 
-NOT (5'-TCAAACTAGGACCGAGTC-3') and
MB (5'-TACCTTGTTACTTCACCCCA-3') and the Taq Mastermix (Qiagen). After PCR product purification using the Qiaquick PCR purification kit (Qiagen), sequencing reactions were performed using the BigDye Terminator sequencing kit (Applied Biosystems) and primers *
, *O, PD and 3 described by Coenye et al. (1999)
. The sequences were determined using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Phylogenetic analysis was done using the program BIONUMERICS (version 3.5; Applied Maths). Pairwise alignment similarities were calculated and a dendrogram was constructed using the neighbour-joining method (Fig. 1
). No correction factor was used. The strains showed sequence similarities of 100 % among each other and were classified within the S. intermedius phylogenetic species group as defined by Takahashi et al. (1999)
. Sequence similarities above 99 % were obtained with the species S. delphini, S. intermedius and S. schleiferi.
|
DNADNA hybridizations were performed between strains LMG 22219T and LMG 22220, and the type strains of S. delphini (DSM 20771T), S. intermedius (LMG 13351T) and S. schleiferi subsp. schleiferi (LMG 13347T). DNA was prepared as described above. The microplate method was used as described by Ezaki et al. (1989)
and Goris et al. (1998)
, using an HTS7000 Bio Assay Reader (Perkin Elmer) for the fluorescence measurements. Biotinylated DNA was hybridized with single-stranded unlabelled DNA, non-covalently bound to microplate wells. Hybridizations were performed at 35 °C in a hybridization mixture containing 2x SSC (0·15 M sodium chloride, 0·015 M sodium citrate), 5x Denhardt's solution (Quantum), 2·5 % dextran sulphate, 50 % formamide, 100 µg denatured salmon sperm DNA ml1 and 1250 ng biotinylated probe DNA ml1. Hybridization levels of 96 % were found between strains LMG 22219T and LMG 22220, which indicates that the strains constitute a single species. Both strains had binding values of 54 % with S. delphini DSM 20771T, 3846 % with S. intermedius LMG 13351T and 1618 % with S. schleiferi subsp. schleiferi LMG 13347T. The latter data indicate that the novel isolates constitute a single species that is distinct from staphylococcal species with validly published names.
Phenotypic tests were applied to the novel isolates. Haemolysis and growth characteristics were tested on Columbia agar base (Oxoid) supplemented with 5 % sheep blood. Coagulase and clumping-factor tests were carried out with rabbit plasma for the detection of staphylocoagulase (bioMérieux). For DNase testing, a standardized plate method was used which discriminates between the strong reactions typical of most coagulase-positive species and the weak reactions often seen with other staphylococci (Devriese & Van de Kerckhove, 1979
). Acid production from carbohydrates was tested in API 50 CH galleries (bioMérieux) under paraffin cover, and other biochemical reactions were tested in API STAPH (bioMérieux) and STAPH-ZYM galleries (Rosco) with the additional susceptibility tests included in this system and acetoin diagnostic test tablets of the same origin. It was observed that alkaline phosphatase reactions were positive in the STAPH-ZYM tests, but negative in the API STAPH tests. Acriflavine MICs were determined as described by Devriese (1981)
. The strains differed from S. aureus subsp. aureus by their lack of pigment, by the absence of clumping-factor activity, by their positive reactions in tests for pyrrolidonyl arylamidase and ONPG (
-galactosidase) and by their sensitivity to 8 µg acriflavine ml1. The acriflavine MICs were in the range 0·81·6 µg ml1 for the four novel isolates, whereas they amounted to 12·5 and 25 µg ml1 for S. aureus strains (Devriese, 1981
). S. aureus strains are unique in being the only staphylococci described, to date, that are resistant to more than 8 µg acriflavine ml1 a level used in the selective isolation of this species in food microbiology (Isigidi et al., 1989
).
The strains are less easy to differentiate from S. intermedius. Many S. intermedius strains have clumping-factor activity and that latter species shows either weak or non-existent acid production from maltose. Erroneous identifications as S. schleiferi subsp. coagulans can be avoided by testing for acidification of trehalose. In addition, S. delphini differs from the novel species in its negative DNase reaction. Methods of differentiation from other coagulase-positive and related species are shown in Table 1
.
Overall, the results of the present study allowed us to assign the four strains LMG 22219T to LMG 22222 to a novel species, for which we propose the name Staphylococcus pseudintermedius sp. nov. A detailed description is given below. The identification of this novel species offers a real challenge to the veterinary diagnostic bacteriologist. These strains are most likely to be confused with S. intermedius. Confirmation of S. pseudintermedius identifications by molecular methods is recommended.
Despite the fact that all four strains upon which the species description is based were isolated from lesions, internal as well as external, in none of the cases was a clear-cut indication of a pathogenic role present.
Description of Staphylococcus pseudintermedius sp. nov.
Staphylococcus pseudintermedius [Gr. adj. pseudes or pseudos false; L. masc. adj. intermedius intermediate, and also a specific epithet; N.L. masc. adj. pseudintermedius a false (Staphylococcus) intermedius, because of the high phenotypic similarity to S. intermedius].
Consists of Gram-positive cocci predominantly arranged in groups. Colonies on Columbia sheep blood agar are non-pigmented and surrounded by double zone haemolysis. The outer band, which is incompletely haemolytic, turns into complete haemolysis after being put at 4 °C (hotcold haemolysis), and is typical of staphylococcal
-haemolysin (a sphingomyelinase). The strains are catalase-positive and coagulate rabbit plasma, but they are clumping-factor-negative in the so-called slide or rapid coagulase test. A strong DNase is produced. Positive in tests for acetoin,
-glucosidase, arginine dihydrolase, urease, nitrate reduction, pyrrolidonyl arylamidase and ONPG (
-galactosidase). Does not produce
-glucuronidase. Susceptible to 8 µg acriflavine ml1 and to novobiocin. Resistant to deferoxamine. Acid is produced from glycerol (weakly and delayed), ribose, galactose, D-glucose, D-fructose, D-mannose, mannitol (weakly and delayed), N-acetylglucosamine, maltose, lactose, sucrose, trehalose and D-turanose (weakly and delayed). No acid is produced from erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, adonitol, methyl
-D-xyloside, L-sorbose, rhamnose, dulcitol, inositol, sorbitol, methyl
-D-glucoside, methyl
-D-xyloside, amygdalin, arbutin, aesculin, salicin, cellobiose, melibiose, inulin, melezitose, D-raffinose, starch, glycogen, xylitol, D-lyxose, D-tagatose, D-fucose, L-fucose, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate. The G+C content of the DNA is 38 mol%.
The type strain is LMG 22219T (=ON 86T=CCUG 49543T). Strains have been isolated from lesions in different animal host species, but the habitat and pathogenic activity are unknown.
| REFERENCES |
|---|
|
|
|---|
Baele, M., Storms, V., Haesebrouck, F., Devriese, L. A., Gillis, M., Verschraegen, G., De Baere, T. & Vaneechoutte, M. (2001). Application and evaluation of the interlaboratory reproducibility of tRNA intergenic length polymorphism analysis (tDNA-PCR) for identification of Streptococcus species. J Clin Microbiol 39, 14361442.
Calvo, J., Hernandez, J. L., Farinas, M. C., Garcia-Palomo, D. & Aguero, J. (2000). Osteomyelitis caused by Staphylococcus schleiferi and evidence of misidentification of this Staphylococcus species by an automated bacterial identification system. J Clin Microbiol 38, 38873889.
Coenye, T., Falsen, E., Vancanneyt, M., Hoste, B., Govan, J. R. W., Kersters, K. & Vandamme, P. (1999). Classification of Alcaligenes faecalis-like isolates from the environment and human clinical samples as Ralstonia gilardii sp. nov. Int J Syst Bacteriol 49, 405413.[CrossRef][Medline]
De La Fuente, R., Suarez, G. & Schleifer, K. H. (1985). Staphylococcus aureus subsp. anaerobius subsp. nov., the causal agent of abscess disease of sheep. Int J Syst Bacteriol 35, 99102.[CrossRef]
Devriese, L. A. (1981). Baird-Parker medium supplemented with acriflavine, polymyxins and sulphonamide for the selective isolation of Staphylococcus aureus from heavily contaminated materials. J Appl Bacteriol 50, 351357.[Medline]
Devriese, L. A. & Hajek, V. (1980). Identification of pathogenic staphylococci isolated from animals and foods derived from animals. J Appl Bacteriol 49, 111.[Medline]
Devriese, L. A. & Van de Kerckhove, A. (1979). A comparison of methods and the validity of deoxyribonuclease tests for the characterization of staphylococci isolated from animals. J Appl Bacteriol 46, 385393.[Medline]
Devriese, L. A., Hajek, V., Oeding, P., Meyer, S. & Schleifer, K. H. (1978). Staphylococcus hyicus (Sompolinsky 1953) comb. nov. and Staphylococcus hyicus subsp. chromogenes subsp. nov. Int J Syst Bacteriol 28, 482490.[CrossRef]
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224229.[CrossRef]
Foster, G., Ross, H. M., Hutson, R. A. & Collins, M. D. (1997). Staphylococcus lutrae sp. nov., a new coagulase-positive species isolated from otters. Int J Syst Bacteriol 47, 724726.[CrossRef][Medline]
Freney, J., Kloos, W. E., Hajek, V., Webster, J. A., Bes, M., Brun, Y. & Vernozy-Rozand, C. (1999). Recommended minimal standards for description of new staphylococcal species. Int J Syst Bacteriol 49, 489502.[CrossRef][Medline]
Goris, J., Suzuki, K., De Vos, P., Nakase, T. & Kersters, K. (1998). Evaluation of a microplate DNADNA hybridization method compared with the initial renaturation method. Can J Microbiol 44, 11481153.[CrossRef]
Hajek, V. (1976). Staphylococcus intermedius, a new species isolated from animals. Int J Syst Bacteriol 26, 401408.[CrossRef]
Hébert, G. A., Crowder, C. G., Hancock, G. A., Jarvis, W. R. & Thornsberry, C. (1988). Characteristics of coagulase-negative staphylococci that help differentiate these species and other members of the family Micrococcaceae. J Clin Microbiol 26, 19391949.
Igimi, S., Takahashi, E. & Mitsuoka, T. (1990). Staphylococcus schleiferi subsp. coagulans subsp. nov., isolated from the external auditory meatus of dogs with external ear otitis. Int J Syst Bacteriol 40, 409411.[CrossRef][Medline]
Isigidi, B. K., Devriese, L. A., Croegaert, T. & Van Hoof, J. (1989). A highly selective two-stage-enumeration method for the selective enumeration of Staphylococcus aureus in foods. J Appl Bacteriol 66, 379384.[Medline]
Kloos, W. E. & Bannerman, T. L. (1999). Staphylococcus and Micrococcus. In Manual of Clinical Microbiology, 7th edn, pp. 264282. Edited by P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover & R. H. Yolken. Washington, DC: American Society for Microbiology.
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159167.
Pitcher, D. G., Saunders, N. A. & Owen, R. J. (1989). Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8, 151156.
Takahashi, E. T., Satoh, I. & Kikuchi, N. (1999). Phylogenetic relationships of 38 taxa of the genus Staphylococcus based on 16S rRNA gene sequence analysis. Int J Syst Bacteriol 49, 725728.[CrossRef][Medline]
Vandenesch, F., Labeau, C., Bes, M. & 7 other authors (1994). Clotting activity in Staphylococcus schleiferi subspecies from human patients. J Clin Microbiol 32, 388392.
Varaldo, P. E., Kilpper-Bälz, R., Biavasco, F., Satta, G. & Schleifer, K. H. (1988). Staphylococcus delphini sp. nov., a coagulase-positive species isolated from dolphins. Int J Syst Bacteriol 38, 436439.[CrossRef]
This article has been cited by other articles:
![]() |
S. Descloux, A. Rossano, and V. Perreten Characterization of New Staphylococcal Cassette Chromosome mec (SCCmec) and Topoisomerase Genes in Fluoroquinolone- and Methicillin-Resistant Staphylococcus pseudintermedius J. Clin. Microbiol., May 1, 2008; 46(5): 1818 - 1823. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. Bannoehr, N. L. Ben Zakour, A. S. Waller, L. Guardabassi, K. L. Thoday, A. H. M. van den Broek, and J. R. Fitzgerald Population Genetic Structure of the Staphylococcus intermedius Group: Insights into agr Diversification and the Emergence of Methicillin-Resistant Strains J. Bacteriol., December 1, 2007; 189(23): 8685 - 8692. [Abstract] [Full Text] [PDF] |
||||
![]() |
T. Sasaki, K. Kikuchi, Y. Tanaka, N. Takahashi, S. Kamata, and K. Hiramatsu Reclassification of Phenotypically Identified Staphylococcus intermedius Strains J. Clin. Microbiol., September 1, 2007; 45(9): 2770 - 2778. [Abstract] [Full Text] [PDF] |
||||
![]() |
S. I. Steen and P. J. Webb Meticillin resistance in veterinary staphylococcal isolates Vet Rec., May 5, 2007; 160(18): 635 - 636. [Full Text] [PDF] |
||||
![]() |
T. Sasaki, K. Kikuchi, Y. Tanaka, N. Takahashi, S. Kamata, and K. Hiramatsu Methicillin-Resistant Staphylococcus pseudintermedius in a Veterinary Teaching Hospital J. Clin. Microbiol., April 1, 2007; 45(4): 1118 - 1125. [Abstract] [Full Text] [PDF] |
||||
![]() |
L. Van Hoovels, A. Vankeerberghen, A. Boel, K. Van Vaerenbergh, and H. De Beenhouwer First Case of Staphylococcus pseudintermedius Infection in a Human J. Clin. Microbiol., December 1, 2006; 44(12): 4609 - 4612. [Abstract] [Full Text] [PDF] |
||||
![]() |
D. A. Bemis, R. D. Jones, L. E. Hiatt, E. D. Ofori, B. W. Rohrbach, L. A. Frank, and S. A. Kania Comparison of Tests To Detect Oxacillin Resistance in Staphylococcus intermedius, Staphylococcus schleiferi, and Staphylococcus aureus Isolates from Canine Hosts. J. Clin. Microbiol., September 1, 2006; 44(9): 3374 - 3376. [Abstract] [Full Text] [PDF] |
||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |