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Elevation of Staphylococcus sciuri subsp. lentus (Kloos et al.) to Species Status: Staphylococcus lentus (Kloos et al.) comb. nov.
System. Appl. Microbiol. 4, 382- 387 (1983)
Lehrstuhl fiir Mikrobiologie, Techn ische Universitat M iinchen, 8000 Miinchcn 2, Federal Republ ic of Germany 2Faculty of Veterinary Medicine, University of Ghent, 9000 Ghent, Belgium
1
Elevation of Staphylo coccus sciuri subsp. lentus (Kloos et al.) to Species Status: Staphylococcus lentus (Kloos et al.) comb. nov. K. H. SCHLEIFER!, U. GEYER!, R. KILPPER-BALZ t, and L. A. DEVRIESE 2
Received M arch 9, 1983
Summary Deoxyribonucleic acid (DNA)-DNA hybridization and physiological studies on 15 strains of S. sciuri subsp. lentus and 4 stra ins of S. sciuri subsp. sciuri demonstrated that specie; rank should be conferred on these subspecies. Strain ATee 29070, the type strain of S. sciuri subsp. lentils, is proposed as type strain of S. lentils. An emended description of this strain is given. Key words : Staphylococcus sciuri, S.lentus - Taxonomy - Classification
Introduction
Kloos et al. (1976) p rop osed the n ame Staphylococcus sciuri for a gro up of staphylo cocci that contain ed a unique p eptidoglycan t ype , Lys-Ala-C ly., and w ere previously pl aced in Staphylococcus group III by Schleifer and Kocur (1973). The major h ab it at of S. sciuri is anima l skin; it is very rarely fo und on h uman skin. Currently, t wo subspecies a re recognized : S. sciuri subsp. sciuri and S. sciuri subsp. lentus. S. sciuri subsp. sciuri is commonly isolated from the skin of rodents and somewhat less frequently from the skin of other mammals and marsupial s. S. sciuri subsp . lentus is often found on the skin an d udders of goat s and sheep. The prop osal t o di vid e th e species S. sciuri in to two su bspecies was so me wha t arbitrary an d ba sed primarily on th e un ique cha racteristics th at are sha red by th ese staphylococci (Kloos et al., 1976). Pr elim inar y DNA-D NA hybridizati on studies between a few st rains of S. sciuri subsp. sciuri and S. sciuri subsp . lentus (Meyer, 1979; Kloos, 1980) sugges ted that these subsp ecies may we ll represent sep ar at e spe cies. In th e present study th e DNA relat edness of th e type strains and so me additio na l strains of S. sciuri subsp, sciuri and S. sciuri subsp. lentus has been determined. In addition , the peptidoglycan type and the oxidase re action have been re-examined.
Elevation of S. sciuri subsp. lentus to Species Status
383
Material and Methods The organisms studied are listed in Table 1. The strains were originally isolated from goat milk (ATCC 29070, K14, K1S, K17, K18, K19, K20), pig skin (VA301, VA609), nares of chicken (VIlIS, P4) and soy bean oil meal (Bucher 313, 606). Culture conditions were similar to those described previously (Kloos et al., 1974 ;1976). Tryptic Soy Agar (Difco, Detroit, USA) was used additionally. Procedures for the preparation of cell walls and determination of peptidoglycan type have also been described previously (Schleifer and Kandler, 1967; 1972).Acid production from carbohydrates was tested in Phenol Red Broth (Gibco, Paisley, Scotland) with 1% sugars added from filter-sterilised solutions. Urease was detected on Christensen Urea Agar (Oxford, Basingstoke, U.K.). Staphylokinase activity was tested on bovine fibrin plates with dog serum as a plasminogen source. Plates without plasminogen were used as controls (Devriese and Van de Kerckhove, 1980). The oxidase reaction was determined using the technique proposed by Faller and Schleifer (1981). The determination of the cytochrome pattern was performed as previously reported (Faller et al., 1980). Isolation of DNA and DNA-DNA hybridization experiments were carried out as described previously (Schleifer et al., 1979; Kilpper et al., 1980). Results and Discussion
1. Colony characteristics Growth was better on Trypticase Soy Agar (TSA) than on P agar. Three strains (CCM 2598, K14 and K20) produced mucoid colonies on these 2 media. They showed partially confluent growth and were white. The non-mucoid strains produced colonies which were 2 to 7 mm in diameter after 5 days on TSA or 0.5 to 5 mm on P agar. They had entire edges and were smooth with glistening surfaces, convex, opaque, white, gray-white or creamy.
2. Peptidoglycan type and oxidase reaction All strains of S. sciuri supbsp. sciuri and S. sciuri subsp. lentus contain the peptidoglycan type Lys-Ala-Gly, that is characteristic for them (Table 1). The same peptidoglycan type is also found in the two strains of uncertain relationship. As already mentioned previously, small amounts of glycine (0.1-0.4 mol/mol glutamic acid) can be replaced by L-serine (Kloos et al., 1976). Previous studies demonstrated that strains of S. sciuri subsp. sciuri and S. sciuri subsp. lentus exhibit two c-type cytochromes (Faller et al., 1980) whereas staphylococci usually (exception: S. caseolyticus, Schleifer et al., 1982) lack c-type cytochromes. The presence of these cytochromes can easily be detected by applying the modified oxidase test of Faller and Schleifer (1981). All strains of S. sciuri subsp. sciuri and S. sciuri subsp. lentus investigated in the present study were oxidase positive. Some of the strains, however, exhibited only a weak positive reaction. The cytochrome pattern of these strains was examined. All of them possess two c-type cytochromes with the maxima of their a-peaks at 549 nm and 554 nm. However, the cytochrome c content of these strains was much lower (about one fifth) than that of strains revealing a strong oxidase positive reaction.
3. DNA-DNA hybridization Results of the DNA-DNA hybridization studies clearly demonstrate that the two subspecies should be considered as separate species. Reassociation reactions carried
384
K.H. Schleifer, U. Geyer, R. Kilpper-Balz, and L. A. Devriese
Table 1. Origin of strains studied, their peptidoglycan type and oxidase reaction Species
Origin and number of strains
Peptidoglycan type
Oxidase reaction
S. sciuri subsp. sciuri
ATCC 29062T Bucher 512 Kloos SC 226 SC 231
Lys-Ala -Gly, Lys-Ala-Gly, Lys-Ala-Gly, (Ser)> Lys-Ala-GlY4 (Ser)>
S. sciuri subsp. lentus
ATCC 29070T Bucher 313 Bucher 606 CCM 2436 CCM 2598 Devriese VA301 Devriese VA609 Devriese VIII5 Devriese P4 Roguinsky K14 K15 K17 K18 K19 K20
Lys-Ala-Gly, Lys-Ala-Gly, Lys-Ala-Gly, Lys-Ala-Gly, Lys-Ala-Gly, Lys-Ala-Gly, Lys-Ala-Gly, Lys-Ala-Gly, Lys-A la-Gly, Lys-Al a-Gly, Lys-Ala-Gly, Lys-Ala-Gly, Lys-Ala-Gly, Lys-Ala-Gly, Lys-Ala-Gly,
Strains of uncertain relationship
CCM 2611 CCM 2614
Lys-Ala-Gly, Lys-Ala-Gly,
+ + + + + + + + + + + + + + + (+)b (+)b (+)b + + +
small amounts of glycine are replaced by L-serine. weak positive. ATCC, American Type Culture Collection , Rockville, MD; Bucher, Dr. E. Bucher, Bay. Landesanstalt fur Bodenkultur und Pflanzenbau, Miinchen ; CCM, Czechoslovac Culture Collection, Brno, CSSR; Devriese, Dr. L.Devriese, Faculty of Vet. Med., Ghent, Belgium; Kloos, Dr. W.Kloos, Nort h Carolina State University, Raleigh, USA; Roguinsky, Dr. M.Roguinsky, Nouzilly, France. a
b
out under optimal conditions between DNAs of strains of these two subspecies revealed homology va lues of 45% or less (Table 2), whereas within one subspecies DNA homology values were 80-100%. The DN A- DN A homology values between the t wo subspecies are even lower (25% or less) by applying stringent reassociation conditions (Meyer, 1979 ; Kloos, 1980). The t wo subsp ecies arc more closely related to one another than to other staphylococci but they are genetically sufficient distinct from each other to w arran t a sepa rate species status. T his is also supported by the immuno logical relatedn ess of th eir fructose-L e-biphosphate aldolases (Fischer et al. , 1983). The aldolas es of S. sciuri subsp. sciuri and S. sciuri subps. lentus gave a cross-reaction with an immunological distance of 67. The immunological d istan ces for the other reference species were higher than 100. The two strains of uncertain relationship (CCM 2611, 2614) are distinct from both S. sciuri subsp. lentus an d S. sciuri subsp. sciuri. Further studies with a larger number of strains are needed to resolve the taxonomic status of these strains. We propose to consider S. sciuri subsp. lentus as separate species, Staphylococcus lentus (Kloos, Schleifer, Smith, 1976) comb. nov. The following description of S. lentus is based on 15 phenotypically and genotypically well characterized strains.
Elevation of S. sciuri subsp . lentil s to Species Status
385
Table 2. DNA-DNA hybrid ization values among various strains of S. sciuri subsp. sciuri, S. sdu ri subsp. lentils and some related staphylococci" Sour ce of filter-bound DNA
S. sciuri subsp. sciuri ATCC 29062T Bucher 512 Kloos SC226 Kloos SC231
% relative bind ing of labeled DNA from S. sciuri subsp. sciuri S. sciuri subsp. lentils ATCC 29062T ATCC 29070T 100 90 88 89
S. sciuri subsp. lentil s ATCC 29070T Bucher 313 Bucher 606 CCM 2436 CCM 2598 Dcvriese VA301 Devriese VA609 Devriese VIII5 Devr iese P4 Rogu insky K14 K15 K17 K18 K19 K20
38 40 36 41 43 40 39 42 41 38 42 n.d . n.d. n.d. 45
Strains of uncertain relationship CCM 2611 CCM 2614
53 42
36 35 41 39 100 86 85 94 86 83 85 87 86 80 84 87 94 96 100 44 39
a Optimal reassociation cond itions were emplo yed corre sponding to approximately 25 DC below the thermal melting point of DNA (0.45 M NaCi plus 0.045 M sodium citrate adjusted to 10% formam ide, 60 °C). T T ype strain (Kloos et al., 1976).
Staphylococcus lentus (Kloos , Schleifer, Smith, 1976) comb. nov. (Staphylococcus sciuri subsp. lentus, Kloos , Schleifer and Smith, 1976, 30). len'tus. L. adj. lentus slow; pertaining to slow growth. Cocci, 0.7 to 1.2 f-l m in
diameter, occurring in tetrads, pairs and singly. Gram positive. Non-motile and non-sporeforming. Colonies were usually 2-7 mm in diameter. They were glistening to wet-glossy in appearance, opaque and convex. They were usually gray-white to white and less frequently creamy. Some strains produced mucoid colonies. Facultative anaerobe s. Growth was much better under aerobic condit ions. Glucose was only weakly degraded under anaerobic conditions. Therefore, these organisms can be misclassified as micrococci on the basis of the oxidation/fermentation (O/F)test . Growth occurred very slowly at NaCI concentrations up to 10% . The opt imal growth temperature ran ge was 25 to 35°C. Only poor or no growth occurred at 15°C or 45°C. All strain s were catalase-positive, reduced nitr ate and demonstrat-
386
K.H.Schleifer, U.Geyer, Ri Kilpper-Balz, and L.A. Devriese
Table 3. Useful properties in the differentiation of novobiocin-resistant staphylococci Species
S.lentus
s. sciuri
S. xylosus
S. saprophyticus
S. cohnii
S. gallinarum
Lys-Gly,_.
Lys-Gly ., Ser
Lys-Gly,_.
Lys-Gly" Ser
D
(+)
D
+
D D
+
D
+
+
D
+ +
+
Properties Lys-Ala-Gly, Lys-Ala-Gly, Peptidoglycan type +a Oxidase test + (cytochrome c) Anaerobic growth (+) (thioglycolate) Nitrate reduction + + Acetoin production D Staphylokinase Urease Aerobically acid from Sucrose + + D Xylose + Cellobiose + + D Arabinose + Raffinose + Salicin + + Melibiose D+
D+
+
+ + + D+ + + D+ +
+,
a Symbols: 90% or more strains positive; -, 90% or more strains negative; D, > 10 < 90% strains positive; D+, > 75 < 90% strains positive; (+), delayed reaction.
ed weak or no phosphatase activity. All strains failed to produce coagulase and to exhibit hemolysin activity. Acetoin was usually not produced. Urease was negative. Many strains produced staphylokinase (VIlIS, VA301, Bucher 606, CCM 2611 and CCM 2614 were staphylokinase-negative). All strains were oxidase positive when using the modified oxidase test described by Faller and Schleifer (1981). Acid was produced aerobically from a wide range of carbohydrates. All strains produced acid aerobically from glucose, fructose, ribose, cellobiose, mannitol, mannose, maltose, galactose, sucrose, lactose, trehalose, salicin, gentiobiose, arabinose, glycerol, esculin and arbutin. Most reacted positive in tests with raffinose (only CCM 2611 was negative), melibiose (Bucher 313, K19 and CCM 2611 were negative) and sorbitol (Bucher 313 and K1S were negative). Many strains produce weakly acid from fucose (ATCC 29070, Bucher 313, K1S, K17, K19, CCM 2611, P4, VIlIS, VA301 and VA609) and from rhamnose (VIlIS, VA301, CCM 2S98, K14, K1S, K17, K19, K20). All were melizitose-, arabitol- and xylitol-negative. The strains were slightly resistant to novobiocin (minimal inhibitory concentrations 2 to 4 ,Ltg/ml). All strains studied contained peptidoglycan of Lys-Ala-Gly, type. The G + C contents of the DNAs of three strains was found to vary between 29.8 and 34.2 mol% (Kloos et aI., 1976). DNA-DNA hybridization studies proved that strains of S. lentus are closely related to one another (Table 2). The main characters for the differentiation of S. lentus and S. sciuri from each other and other novobiocin-resistant coagulasenegative staphylococci are listed in Table 3. S. lentus can be distinguished from these staphylococci primarily on the basis of the peptidoglycan type, oxidase reactionand the aerobic acid production from a wide variety of carbohydrates.
Elevation of S. sciuri subsp. lentus to Species Status
387
Strain ATCC 29070 (originally designated Roguinsky K21) isolated from the udder of goat was proposed by Kloos et a1. (1976) as type strain of S. sciuri subsp. lentus and is now the type strain of S. lentus. A detailed description of this type strain is given by Kloos et a1. (1976). The following emendation can be made: Strain ATCC 29070 is oxidase positive (Faller and Schleifer, 1981), urease-negative, staphylokinase-positive and contains c-type cytochromes. The strain is weakly maltose- and melibiose-positive in phenol red broth. References Devriese, L. A., van de Kerckhove, A.: A comparison of methods used for testing staphylokinase (fibrinolysin) production is Staphylococcus strains. Antonie v. Leeuwenhoek 46, 457-465 (1980) Faller, A. H., Gotz, F., Schleifer, K. H.: Cytochrome-patterns of staphylococci and micrococci and their taxonomic implications. Zbl. Bakt. Hyg., 1.Abt, Orig. C 1, 26-39 (1980) Faller, A. H., Schleifer, K. H.: Modified oxidase and benzidine tests for separation of staphylococci from micrococci. J. Clin. Microbiol. 13, 1031-1035 (1981) Fischer, S., Tsugita, A., Kreutz, B., Schleifer, K. H.: Immunochemical and proteinchemical studies of class I fructose-Le-biphosphate aldolases from staphylococci. Int. J. system. Bact. 33, in press Kilpper, R., Buhl, V., Schleifer, K. H.: Nucleic acid homology studies between Peptococcus saccharolyticus and various anaerobic and facultative anaerobic Gram-positive cocci. FEMS Microbiol. Lett. 8, 205-210 (1980) Kloos, W. E.: Natural populations of the genus Staphylococcus. Ann. Rev. Microbiol. 34, 559-592 (1980) Kloos, W. E., Tornabene, T. G., Schleifer, K. H.: Isolation and characterization of micrococci from human skin, including two new species: Micrococcus lylae and Micrococcus kristinae. Int. J. system. Bact. 24, 29-101 (1974) Kloos, W.E., Schleifer, K.H., Smith, R.F.: Characterization of Staphylococcus sciuri sp. nov. and its subspecies. Int. J. system. Bact. 26, 22-37 (1976) Meyer, S. A.: Nucleic acid homology within the genus Staphylococcus. Doctoral Thesis, Techn. Univ. Munich (1979) Schleifer, K. H., Kandler, 0.: Zur chemischen Zusammensetzung der Zellwand der Streptokokken. 1. Die Arninosauresequenz des Mureins von Str. thermophilus and Str. faecalis. Arch. Mikrobiol. 57, 335-364 (1967) Schleifer, K. H., Kandler, 0.: Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bact. Rev. 36, 407-477 (1972) Schleifer, K. H., Kocur, M.: Classification of staphylococci based on chemical and biochemical properties. Arch. Mikrobiol. 93, 65-85 (1973) Schleifer, K. H., Meyer, S. A., Rupprecht, M.: Relatedness among coagulase-negative staphylococci: Deoxyribonucleic acid reassociation and comparative immunological studies. Arch. Microbiol. 122, 93-101 (1979) Schleifer, K. H., Kilpper-Bdlz, R., Fischer, V., Faller, A., Endl, ].: Identification of "Micrococcus candidus" ATCC 14852 as a strain of Staphylococcus epidermidis and of "Micrococcus caseolyticus" ATCC 13548 and Micrococcus varians ATCC 29750 as members of a new species, Staphylococcus caseolyticus. Int. J. system. Bact. 32, 15-20 (1982)
Professor Dr. K. H. Schleifer, Technische Universitat Miinchen, Lehrstuhl fur Mikrobiologie, Arcisstr. 21, D-8000 Miinchen 2, FRG
Lehrstuhl fiir Mikrobiologie, Techn ische Universitat M iinchen, 8000 Miinchcn 2, Federal Republ ic of Germany 2Faculty of Veterinary Medicine, University of Ghent, 9000 Ghent, Belgium
1
Elevation of Staphylo coccus sciuri subsp. lentus (Kloos et al.) to Species Status: Staphylococcus lentus (Kloos et al.) comb. nov. K. H. SCHLEIFER!, U. GEYER!, R. KILPPER-BALZ t, and L. A. DEVRIESE 2
Received M arch 9, 1983
Summary Deoxyribonucleic acid (DNA)-DNA hybridization and physiological studies on 15 strains of S. sciuri subsp. lentus and 4 stra ins of S. sciuri subsp. sciuri demonstrated that specie; rank should be conferred on these subspecies. Strain ATee 29070, the type strain of S. sciuri subsp. lentils, is proposed as type strain of S. lentils. An emended description of this strain is given. Key words : Staphylococcus sciuri, S.lentus - Taxonomy - Classification
Introduction
Kloos et al. (1976) p rop osed the n ame Staphylococcus sciuri for a gro up of staphylo cocci that contain ed a unique p eptidoglycan t ype , Lys-Ala-C ly., and w ere previously pl aced in Staphylococcus group III by Schleifer and Kocur (1973). The major h ab it at of S. sciuri is anima l skin; it is very rarely fo und on h uman skin. Currently, t wo subspecies a re recognized : S. sciuri subsp. sciuri and S. sciuri subsp. lentus. S. sciuri subsp. sciuri is commonly isolated from the skin of rodents and somewhat less frequently from the skin of other mammals and marsupial s. S. sciuri subsp . lentus is often found on the skin an d udders of goat s and sheep. The prop osal t o di vid e th e species S. sciuri in to two su bspecies was so me wha t arbitrary an d ba sed primarily on th e un ique cha racteristics th at are sha red by th ese staphylococci (Kloos et al., 1976). Pr elim inar y DNA-D NA hybridizati on studies between a few st rains of S. sciuri subsp. sciuri and S. sciuri subsp . lentus (Meyer, 1979; Kloos, 1980) sugges ted that these subsp ecies may we ll represent sep ar at e spe cies. In th e present study th e DNA relat edness of th e type strains and so me additio na l strains of S. sciuri subsp, sciuri and S. sciuri subsp. lentus has been determined. In addition , the peptidoglycan type and the oxidase re action have been re-examined.
Elevation of S. sciuri subsp. lentus to Species Status
383
Material and Methods The organisms studied are listed in Table 1. The strains were originally isolated from goat milk (ATCC 29070, K14, K1S, K17, K18, K19, K20), pig skin (VA301, VA609), nares of chicken (VIlIS, P4) and soy bean oil meal (Bucher 313, 606). Culture conditions were similar to those described previously (Kloos et al., 1974 ;1976). Tryptic Soy Agar (Difco, Detroit, USA) was used additionally. Procedures for the preparation of cell walls and determination of peptidoglycan type have also been described previously (Schleifer and Kandler, 1967; 1972).Acid production from carbohydrates was tested in Phenol Red Broth (Gibco, Paisley, Scotland) with 1% sugars added from filter-sterilised solutions. Urease was detected on Christensen Urea Agar (Oxford, Basingstoke, U.K.). Staphylokinase activity was tested on bovine fibrin plates with dog serum as a plasminogen source. Plates without plasminogen were used as controls (Devriese and Van de Kerckhove, 1980). The oxidase reaction was determined using the technique proposed by Faller and Schleifer (1981). The determination of the cytochrome pattern was performed as previously reported (Faller et al., 1980). Isolation of DNA and DNA-DNA hybridization experiments were carried out as described previously (Schleifer et al., 1979; Kilpper et al., 1980). Results and Discussion
1. Colony characteristics Growth was better on Trypticase Soy Agar (TSA) than on P agar. Three strains (CCM 2598, K14 and K20) produced mucoid colonies on these 2 media. They showed partially confluent growth and were white. The non-mucoid strains produced colonies which were 2 to 7 mm in diameter after 5 days on TSA or 0.5 to 5 mm on P agar. They had entire edges and were smooth with glistening surfaces, convex, opaque, white, gray-white or creamy.
2. Peptidoglycan type and oxidase reaction All strains of S. sciuri supbsp. sciuri and S. sciuri subsp. lentus contain the peptidoglycan type Lys-Ala-Gly, that is characteristic for them (Table 1). The same peptidoglycan type is also found in the two strains of uncertain relationship. As already mentioned previously, small amounts of glycine (0.1-0.4 mol/mol glutamic acid) can be replaced by L-serine (Kloos et al., 1976). Previous studies demonstrated that strains of S. sciuri subsp. sciuri and S. sciuri subsp. lentus exhibit two c-type cytochromes (Faller et al., 1980) whereas staphylococci usually (exception: S. caseolyticus, Schleifer et al., 1982) lack c-type cytochromes. The presence of these cytochromes can easily be detected by applying the modified oxidase test of Faller and Schleifer (1981). All strains of S. sciuri subsp. sciuri and S. sciuri subsp. lentus investigated in the present study were oxidase positive. Some of the strains, however, exhibited only a weak positive reaction. The cytochrome pattern of these strains was examined. All of them possess two c-type cytochromes with the maxima of their a-peaks at 549 nm and 554 nm. However, the cytochrome c content of these strains was much lower (about one fifth) than that of strains revealing a strong oxidase positive reaction.
3. DNA-DNA hybridization Results of the DNA-DNA hybridization studies clearly demonstrate that the two subspecies should be considered as separate species. Reassociation reactions carried
384
K.H. Schleifer, U. Geyer, R. Kilpper-Balz, and L. A. Devriese
Table 1. Origin of strains studied, their peptidoglycan type and oxidase reaction Species
Origin and number of strains
Peptidoglycan type
Oxidase reaction
S. sciuri subsp. sciuri
ATCC 29062T Bucher 512 Kloos SC 226 SC 231
Lys-Ala -Gly, Lys-Ala-Gly, Lys-Ala-Gly, (Ser)> Lys-Ala-GlY4 (Ser)>
S. sciuri subsp. lentus
ATCC 29070T Bucher 313 Bucher 606 CCM 2436 CCM 2598 Devriese VA301 Devriese VA609 Devriese VIII5 Devriese P4 Roguinsky K14 K15 K17 K18 K19 K20
Lys-Ala-Gly, Lys-Ala-Gly, Lys-Ala-Gly, Lys-Ala-Gly, Lys-Ala-Gly, Lys-Ala-Gly, Lys-Ala-Gly, Lys-Ala-Gly, Lys-A la-Gly, Lys-Al a-Gly, Lys-Ala-Gly, Lys-Ala-Gly, Lys-Ala-Gly, Lys-Ala-Gly, Lys-Ala-Gly,
Strains of uncertain relationship
CCM 2611 CCM 2614
Lys-Ala-Gly, Lys-Ala-Gly,
+ + + + + + + + + + + + + + + (+)b (+)b (+)b + + +
small amounts of glycine are replaced by L-serine. weak positive. ATCC, American Type Culture Collection , Rockville, MD; Bucher, Dr. E. Bucher, Bay. Landesanstalt fur Bodenkultur und Pflanzenbau, Miinchen ; CCM, Czechoslovac Culture Collection, Brno, CSSR; Devriese, Dr. L.Devriese, Faculty of Vet. Med., Ghent, Belgium; Kloos, Dr. W.Kloos, Nort h Carolina State University, Raleigh, USA; Roguinsky, Dr. M.Roguinsky, Nouzilly, France. a
b
out under optimal conditions between DNAs of strains of these two subspecies revealed homology va lues of 45% or less (Table 2), whereas within one subspecies DNA homology values were 80-100%. The DN A- DN A homology values between the t wo subspecies are even lower (25% or less) by applying stringent reassociation conditions (Meyer, 1979 ; Kloos, 1980). The t wo subsp ecies arc more closely related to one another than to other staphylococci but they are genetically sufficient distinct from each other to w arran t a sepa rate species status. T his is also supported by the immuno logical relatedn ess of th eir fructose-L e-biphosphate aldolases (Fischer et al. , 1983). The aldolas es of S. sciuri subsp. sciuri and S. sciuri subps. lentus gave a cross-reaction with an immunological distance of 67. The immunological d istan ces for the other reference species were higher than 100. The two strains of uncertain relationship (CCM 2611, 2614) are distinct from both S. sciuri subsp. lentus an d S. sciuri subsp. sciuri. Further studies with a larger number of strains are needed to resolve the taxonomic status of these strains. We propose to consider S. sciuri subsp. lentus as separate species, Staphylococcus lentus (Kloos, Schleifer, Smith, 1976) comb. nov. The following description of S. lentus is based on 15 phenotypically and genotypically well characterized strains.
Elevation of S. sciuri subsp . lentil s to Species Status
385
Table 2. DNA-DNA hybrid ization values among various strains of S. sciuri subsp. sciuri, S. sdu ri subsp. lentils and some related staphylococci" Sour ce of filter-bound DNA
S. sciuri subsp. sciuri ATCC 29062T Bucher 512 Kloos SC226 Kloos SC231
% relative bind ing of labeled DNA from S. sciuri subsp. sciuri S. sciuri subsp. lentils ATCC 29062T ATCC 29070T 100 90 88 89
S. sciuri subsp. lentil s ATCC 29070T Bucher 313 Bucher 606 CCM 2436 CCM 2598 Dcvriese VA301 Devriese VA609 Devriese VIII5 Devr iese P4 Rogu insky K14 K15 K17 K18 K19 K20
38 40 36 41 43 40 39 42 41 38 42 n.d . n.d. n.d. 45
Strains of uncertain relationship CCM 2611 CCM 2614
53 42
36 35 41 39 100 86 85 94 86 83 85 87 86 80 84 87 94 96 100 44 39
a Optimal reassociation cond itions were emplo yed corre sponding to approximately 25 DC below the thermal melting point of DNA (0.45 M NaCi plus 0.045 M sodium citrate adjusted to 10% formam ide, 60 °C). T T ype strain (Kloos et al., 1976).
Staphylococcus lentus (Kloos , Schleifer, Smith, 1976) comb. nov. (Staphylococcus sciuri subsp. lentus, Kloos , Schleifer and Smith, 1976, 30). len'tus. L. adj. lentus slow; pertaining to slow growth. Cocci, 0.7 to 1.2 f-l m in
diameter, occurring in tetrads, pairs and singly. Gram positive. Non-motile and non-sporeforming. Colonies were usually 2-7 mm in diameter. They were glistening to wet-glossy in appearance, opaque and convex. They were usually gray-white to white and less frequently creamy. Some strains produced mucoid colonies. Facultative anaerobe s. Growth was much better under aerobic condit ions. Glucose was only weakly degraded under anaerobic conditions. Therefore, these organisms can be misclassified as micrococci on the basis of the oxidation/fermentation (O/F)test . Growth occurred very slowly at NaCI concentrations up to 10% . The opt imal growth temperature ran ge was 25 to 35°C. Only poor or no growth occurred at 15°C or 45°C. All strain s were catalase-positive, reduced nitr ate and demonstrat-
386
K.H.Schleifer, U.Geyer, Ri Kilpper-Balz, and L.A. Devriese
Table 3. Useful properties in the differentiation of novobiocin-resistant staphylococci Species
S.lentus
s. sciuri
S. xylosus
S. saprophyticus
S. cohnii
S. gallinarum
Lys-Gly,_.
Lys-Gly ., Ser
Lys-Gly,_.
Lys-Gly" Ser
D
(+)
D
+
D D
+
D
+
+
D
+ +
+
Properties Lys-Ala-Gly, Lys-Ala-Gly, Peptidoglycan type +a Oxidase test + (cytochrome c) Anaerobic growth (+) (thioglycolate) Nitrate reduction + + Acetoin production D Staphylokinase Urease Aerobically acid from Sucrose + + D Xylose + Cellobiose + + D Arabinose + Raffinose + Salicin + + Melibiose D+
D+
+
+ + + D+ + + D+ +
+,
a Symbols: 90% or more strains positive; -, 90% or more strains negative; D, > 10 < 90% strains positive; D+, > 75 < 90% strains positive; (+), delayed reaction.
ed weak or no phosphatase activity. All strains failed to produce coagulase and to exhibit hemolysin activity. Acetoin was usually not produced. Urease was negative. Many strains produced staphylokinase (VIlIS, VA301, Bucher 606, CCM 2611 and CCM 2614 were staphylokinase-negative). All strains were oxidase positive when using the modified oxidase test described by Faller and Schleifer (1981). Acid was produced aerobically from a wide range of carbohydrates. All strains produced acid aerobically from glucose, fructose, ribose, cellobiose, mannitol, mannose, maltose, galactose, sucrose, lactose, trehalose, salicin, gentiobiose, arabinose, glycerol, esculin and arbutin. Most reacted positive in tests with raffinose (only CCM 2611 was negative), melibiose (Bucher 313, K19 and CCM 2611 were negative) and sorbitol (Bucher 313 and K1S were negative). Many strains produce weakly acid from fucose (ATCC 29070, Bucher 313, K1S, K17, K19, CCM 2611, P4, VIlIS, VA301 and VA609) and from rhamnose (VIlIS, VA301, CCM 2S98, K14, K1S, K17, K19, K20). All were melizitose-, arabitol- and xylitol-negative. The strains were slightly resistant to novobiocin (minimal inhibitory concentrations 2 to 4 ,Ltg/ml). All strains studied contained peptidoglycan of Lys-Ala-Gly, type. The G + C contents of the DNAs of three strains was found to vary between 29.8 and 34.2 mol% (Kloos et aI., 1976). DNA-DNA hybridization studies proved that strains of S. lentus are closely related to one another (Table 2). The main characters for the differentiation of S. lentus and S. sciuri from each other and other novobiocin-resistant coagulasenegative staphylococci are listed in Table 3. S. lentus can be distinguished from these staphylococci primarily on the basis of the peptidoglycan type, oxidase reactionand the aerobic acid production from a wide variety of carbohydrates.
Elevation of S. sciuri subsp. lentus to Species Status
387
Strain ATCC 29070 (originally designated Roguinsky K21) isolated from the udder of goat was proposed by Kloos et a1. (1976) as type strain of S. sciuri subsp. lentus and is now the type strain of S. lentus. A detailed description of this type strain is given by Kloos et a1. (1976). The following emendation can be made: Strain ATCC 29070 is oxidase positive (Faller and Schleifer, 1981), urease-negative, staphylokinase-positive and contains c-type cytochromes. The strain is weakly maltose- and melibiose-positive in phenol red broth. References Devriese, L. A., van de Kerckhove, A.: A comparison of methods used for testing staphylokinase (fibrinolysin) production is Staphylococcus strains. Antonie v. Leeuwenhoek 46, 457-465 (1980) Faller, A. H., Gotz, F., Schleifer, K. H.: Cytochrome-patterns of staphylococci and micrococci and their taxonomic implications. Zbl. Bakt. Hyg., 1.Abt, Orig. C 1, 26-39 (1980) Faller, A. H., Schleifer, K. H.: Modified oxidase and benzidine tests for separation of staphylococci from micrococci. J. Clin. Microbiol. 13, 1031-1035 (1981) Fischer, S., Tsugita, A., Kreutz, B., Schleifer, K. H.: Immunochemical and proteinchemical studies of class I fructose-Le-biphosphate aldolases from staphylococci. Int. J. system. Bact. 33, in press Kilpper, R., Buhl, V., Schleifer, K. H.: Nucleic acid homology studies between Peptococcus saccharolyticus and various anaerobic and facultative anaerobic Gram-positive cocci. FEMS Microbiol. Lett. 8, 205-210 (1980) Kloos, W. E.: Natural populations of the genus Staphylococcus. Ann. Rev. Microbiol. 34, 559-592 (1980) Kloos, W. E., Tornabene, T. G., Schleifer, K. H.: Isolation and characterization of micrococci from human skin, including two new species: Micrococcus lylae and Micrococcus kristinae. Int. J. system. Bact. 24, 29-101 (1974) Kloos, W.E., Schleifer, K.H., Smith, R.F.: Characterization of Staphylococcus sciuri sp. nov. and its subspecies. Int. J. system. Bact. 26, 22-37 (1976) Meyer, S. A.: Nucleic acid homology within the genus Staphylococcus. Doctoral Thesis, Techn. Univ. Munich (1979) Schleifer, K. H., Kandler, 0.: Zur chemischen Zusammensetzung der Zellwand der Streptokokken. 1. Die Arninosauresequenz des Mureins von Str. thermophilus and Str. faecalis. Arch. Mikrobiol. 57, 335-364 (1967) Schleifer, K. H., Kandler, 0.: Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bact. Rev. 36, 407-477 (1972) Schleifer, K. H., Kocur, M.: Classification of staphylococci based on chemical and biochemical properties. Arch. Mikrobiol. 93, 65-85 (1973) Schleifer, K. H., Meyer, S. A., Rupprecht, M.: Relatedness among coagulase-negative staphylococci: Deoxyribonucleic acid reassociation and comparative immunological studies. Arch. Microbiol. 122, 93-101 (1979) Schleifer, K. H., Kilpper-Bdlz, R., Fischer, V., Faller, A., Endl, ].: Identification of "Micrococcus candidus" ATCC 14852 as a strain of Staphylococcus epidermidis and of "Micrococcus caseolyticus" ATCC 13548 and Micrococcus varians ATCC 29750 as members of a new species, Staphylococcus caseolyticus. Int. J. system. Bact. 32, 15-20 (1982)
Professor Dr. K. H. Schleifer, Technische Universitat Miinchen, Lehrstuhl fur Mikrobiologie, Arcisstr. 21, D-8000 Miinchen 2, FRG