Staphylococcus capitis subsp. ureolyticus subsp. nov. from Human

Vol. 41, No. 1
INTERNATIONAL
JOURNAL OF SYSTEMATIC
BACTERIOLOGY,
Jan. 1991, p. 144-147
0020-7713/91/010144-04$02.0010
Copyright 0 1991, International Union of Microbiological Societies
Staphylococcus capitis subsp. ureolyticus subsp. nov.
from Human Skin
TAMMY L. BANNERMAN’* AND WESLEY E. KLOOS2
Departments of Microbiology’ and Genetics,2 North Carolina State University,
Raleigh, North Carolina 27695-7614
A new subspecies, Staphylococcus cupitis subsp. ureolyticus, was isolated from human skin and is described
on the basis of studies of 15 to 26 strains. DNA-DNA reassociation reactions demonstrated that these strains
were closely related to Stuphylococcus cupitis but were significantly divergent. The strains of S. cupitis subsp.
ureolyticus can be distinguished from S. capitis by their positive urease activity, their ability to produce acid
from maltose under aerobic conditions, their fatty acid profile, and their colony morphology. The type strain
of the new subspecies is strain ATCC 49326.
N-acetylglucosamine were determined by using commercial
rapid-identification system ATB 32 Staph (API System, La
Balme-les-Grottes, France). Pyrrolidonyl arylamidase activity and esculin hydrolysis were determined by using ATB 32
Staph and Baxter-Microscan Pos Combo Type 5 panels
(Baxter Healthcare Corp., Microscan Div., West Sacramento, Calif.). Clumping factor was determined by using a
conventional method (6) and by using the commercially
available Staphaurex test (Wellcome Diagnostics, Dartford,
England). Heat-stable nuclease activity was analyzed by
using thermonuclease agar with toluidine blue from Remel,
Lenexa, Kans., according to the manufacturer’s instructions. Ornithine decarboxylase activity was determined by
using a modification of the test of Moeller (15) as described
in the Manual of Clinical Microbiology, 5th ed. (6), and by
using the ATB 32 Staph test. Alkaline phosphatase, urease,
P-galactosidase, p-glucosidase, and P-glucuronidase activities and arginine utilization were analyzed by using the API
STAPH-IDENT system (Analytab Products, Plainview,
N.Y.). The fatty acid profile was determined by using the
MIDI microbial identification system (model HP5890A; Microbial ID, Inc., Newark, Del.). Cell wall peptidoglycans
were prepared for amino acid analysis as described by
Schleifer and Kandler (16). Preparations were treated for 20
h with 6 N HC14.05% mercaptoethanol at 115°C. The amino
acids of the hydrolysate were quantitatively determined with
a Beckman-Spinco analyzer (model 120B). The strains tested
for cell wall amino acids were S. capitis ATCC 27840T,
Staphylococcus epidermidis ATCC 14990T, Staphylococcus
aureus ATCC 12600T, S . capitis subsp. ureolyticus ATCC
49326T, and S. capitis subsp. ureolyticus ATCC 49325. DNA
base composition was determined by the thermal denaturation method of Marmur and Doty (14). DNA was isolated
and purified by using the procedures of Brenner and coworkers (l),as modified by Kloos and Wolfshohl (12) for
DNA-DNA reassociation reactions. DNA-DNA reassociation reactions were performed and single- and doublestranded DNAs were separated by using the procedures of
Brenner and co-workers (1).
In this paper, we describe a group of staphylococci that
are closely related to Staphylococcus cupitis and constitute
a separate subspecies, Staphylococcus capitis subsp. ureolyticus. S . capitis subsp. ureolyticus was originally considered to be a possible rare biotype of Staphylococcus warneri
or Staphy Zococcus hominis according to the classification
system described by Kloos and Schleifer (8). S. warneri and
S . hominis were similar to this group of organisms in their
colony morphology, in their positive urease activity, in their
ability to produce acid from maltose aerobically, and (in rare
strains) in their ability to produce acid from mannose.
Approximately 12% of the S. horninis strains shared API
STAPH-IDENT profile 2040 with one-half of the S . capitis
subsp. ureolyticus strains tested in this study.
MATERIALS AND METHODS
Bacterial strains. The strains used in this study were
isolated from healthy human volunteers, human clinical
specimens, and animal specimens. The strains isolated from
healthy skin were strains AK 8635, CB 8735, AK 8634, and
CMM 8536 (= ATCC 49324) (from external auditory meatus); AK 8638, NRC 8533,1436-6, DP 4A, and 1375-8 (from
foreheads); AK 8636, DLB 8531, NRC 8535, MAW 843(jT (=
ATCC 49326T) (T = type strain), and JLJ 8524 (from scalps);
CB 8730 (= ATCC 49325), DEM 8530 (= ATCC 49327), and
CMM 8539 (from inguinal areas); MK 8731 (from anterior
nares); and CMM 8544 (from a leg). The human clinical
strains were strains LA 1235 (from necrotic areas of skin);
GA-W, GA-1, and 5717-1 (from blood cultures); 3740-4 (from
a urine culture); and 4147-1 (from a fluid culture). The animal
specimens were strains PAY 1012 (from the chest of a wild
male chimpanzee) and 142.25(a) (from a mixed culture with
Staphylococcus caprae in goat milk). The strains of the other
species included in this study are listed in Table 1.
Character determinations. The following characteristics
were determined as previously described (8, 1&12, 17):
colony morphology and pigment, anaerobic growth in thioglycolate broth, catalase activity, acetylmethylcarbinol (acetoin) production, nitrate reduction, coagulase activity, hemolysis of bovine blood, carbohydrate reactions, and
susceptibility to various antibiotics. The oxidase test was
performed as described by Faller and Schleifer (3). Arginine
arylamidase activity and aerobic acid production from
RESULTS AND DISCUSSION
DNA-DNA reassociation. The DNA relationships between
S . capitis subsp. ureolyticus ATCC 49326T and other strains
belonging to this subspecies and other Staphylococcus species are shown in Table 1.
DNA-DNA reassociation reactions performed under opti-
* Corresponding author.
144
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S. CAPITZS SUBSP. UREOLYTICUS SUBSP. NOV.
VOL. 41. 1991
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TABLE 1. Results of hybridization of staphylococcal DNAs with [methyl-3H]thymidine-labeledDNAs from
S, capitis subsp. ureolyticus, S . capitis, and S . caprae
% Relative binding with labeled DNA from":
S . cupitis subsp.
Source of unlabeled DNA
S . capitis
ATCC 2784QT
ureolyticus
ATCC 49326T
S . caprae
CCM 3573'
Species
Strainb
55°C
70°C
55°C
70°C
55°C
70°C
S . capitis subsp. ureolyticus
ATCC 49326T
ATCC 49324
ATCC 49325
ATCC 49327
DLB 8531
AK 8635
1436-6
MK 8731
142.25(a)
ATCC 27840T
ATCC 27842
WK 819
CCM 3573T
143.11
143.16
143.15
143.14
141.15
ATCC 14990T
ATCC 27836T
ATCC 27844
DSM 20263T
ATCC 43809T
ATCC 12600T
ATCC 33753T
DSM 20260T
CCM 883T
DSM 20266T
ATCC 43957T
ATCC 4395fIT
DSM 20676T
CCM 3572T
ATCC 27848T
MA
CFDD
ATCC 43808T
NCTC 10350T
CBCC 1462T
ATCC 13548T
K-15
ATCC 29062T
100
99
100
98
98
95
91
85
81
88
100
100
97
97
94
95
87
86
89
80
88
91
88
87
90
89
92
83
77
74
78
78
83
82
64
24
100
100
60
21
71
72
69
71
74
66
32
45
31
32
33
35
20
41
30
34
18
19
21
32
13
27
17
17
16
23
14
17
18
23
23
21
19
22
20
13
18
9
8
8
7
7
7
8
7
7
6
6
8
6
7
4
6
100
87
96
25
S . capitis
S . caprae
S.
S.
S.
S.
S.
epidermidis
warneri
horninis
haemolyticus
lugdunensis
S.aureus
S . auricularis
S . cohnii
S . saprophyticus
s.xylosus
S.arlettae
S . equorum
S.kloosii
S . gallinarum
S . simulans
S . carnosus
S . intermedius
S . schleiferi
S . hyicus
S. chromogenes
S.caseolyticus
S.lentus
S . sciuri
63
5
8
6
7
4
a In the homologous reactions, labeled DNAs reassociated with unlabeled DNAs at a level of 93% 2 2%. The percentages of radioactivity bound in the
heterologous reactions were normalized to the percentages of radioactivity in the homologous reactions.
ATCC, Amencan Type Culture Collection, Rockville, Md. ; CCM, Czechoslovak Collection of Microorganisms, J. E. Purkyne University, Brno,
Czechoslovakia; DSM, Deutsche Sammlung von Mikroorganismen, Gottingen, Federal Republic of Germany.
ma1 (55°C) and stringent (70°C) conditions indicated that the
different S. capitis subsp. ureolyticus strains exhibited high
degrees of homology under both conditions (93% k 6% at
55°C and 93% 5 4% at 70°C [mean 5 standard deviation]). S.
capitis ATCC 27840T exhibited high degrees of homology
with some other S . capitis strains (100% at 55°C and 92% 2
5% at 70°C). However, the levels of relatedness between S .
capitis subsp. ureolyticus DNA and S . capitis ATCC 27840T
DNA were significantly lower (89% 5 1%at 55°C and 79% 2
3% at 70°C) and were similar to the levels expected for
separate subspecies.
Further analysis of the levels of DNA relatedness between
S. capitis subsp. ureolyticus ATCC 49326T and other Staphylococcus species indicated that this strain is relatively
closely related to Staphylococcus caprae (71% k 2% at 55°C
and 21% 5 1% at 70°C). The values obtained for the
reciprocal reaction with S. caprae CCM 3573T DNA confirmed this relationship (64% at 55°C and 24% at 70°C). The
significant level of DNA homology between S. caprae andS.
capitis has been mentioned previously (2); it is not surprising
that the level of relatedness between S. capitis subsp.
ureolyticus DNA and S . caprae DNA is rather similar.
Description of Staphylococcus capitis subsp. ureolyticus
subsp. nov. Staphylococcus capitis subsp. ureolyticus (ur.
e.o.ly'ti.cus. N. L. n. urea, urea; Gr. adj. lyticus, dissolving;
N. L. adj. ureolyticus, urea dissolving). The description of S.
capitis subsp. ureolyticus below is based on studies of 15 to
26 strains. Cells are gram-positive cocci that are 0.8 to 1.0
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146
INT. J. SYST.BACTERIOL.
BANNERMAN AND KLOOS
TABLE 2. Variable characteristics of S . capitis subsp.
ureolyticus strains
Characteristic
No. of strains positive/
total no. of strains
tested"
TABLE 3. Fatty acid profiles of S . capitis subsp. ureolyticus and
several related Staphylococcus species
Avg % of total fatty acids in:
% of strains
positiveb
Fatty acid
Colony size of 1 6 mm
Colony pigment
,
Hemolysis
Pyrrolidonyl arylamidase
activity
Acetoin production
Nitrate reduction
Acid produced aerobically from:
D-Mannitol
D-Turanose
D-Melezitose
a-Lactose
Maltose
.
Sucrose
4126
19126
2126 (14/26)
0115 (2/15)
16119 (2/19)
18119 (1119)
84 (11)
95 (5)
25/26 (1/26)
4126 (8126)
0126 (5126)
17/26 (6/26)
25/26 (1126)
25126 (1126)
96 (4)
15 (31)
0 (19)
65 (23)
96 (4)
96 (4)
The values in parentheses are the number of strains showing a weak
positive reactiodtotal number of strains tested.
The numbers in parentheses are the percentage of strains showing a weak
positive reaction.
a
pm in diameter, nonmotile, and nonsporeforming and occur
predominantly in singles, pairs, and clusters. Colonies on P
agar (9) are usually raised, opaque, circular, and glossy and
range in diameter from 4.3 to 7.1 mm (after incubation for 3
days at 35°C and for 2 days at 25°C); 73% have some delayed
yellow pigmentation. The colony surfaces of 75% of the
strains are smooth; 25% of the strains have colonies with
rough surfaces or a granular texture. The colony edges are
entire to slightly irregular. Facultatively anaerobic. Catalase
is produced. Oxidase is not produced. Coagulase, clumping
factor, heat-stable nuclease, alkaline phosphatase, ornithine
decarboxylase, P-glucosidase, P-glucuronidase, P-galactosidase, and arginine arylamidase tests are negative. Urease
positive. Arginine is utilized. No esculin hydrolysis. Acid is
not produced aerobically from D-trehalose, D-xylose, D-cellobiose, L-arabinose, N-acetylglucosamine, and raffinose.
Acid is produced from D-mannose. No anaerobic acid production from D-mannitol. The variable characteristics of S .
cupitis subsp. ureolyticus are shown in Table 2.
The straips from skin are mostly from heads, primarily
ears and foreheads. This habitat distribution is similar to that
of S . cupitis (9). However, unlike S. capitis, during antibiotic
use S . cupitis subsp. ureolyticus increases its range of
habitats to areas other than the head (5).
Fatty acid profile. The results of a fatty acid analysis of S .
cupitis subsp. ureolyticus and closely related members of the
S . epidermidis species group are shown in Table 3.
Compared with S. capitis, the new subspecies has a higher
percentage of iso-C1s:o fatty acid and a lower percentage of
C20:ofatty acid.
Antibiotic susceptibilities. As determined by agar disk
diffusion tests, the strains isolated from the skin of healthy
human volunteers are susceptible to novobiocin, tetracycline, erythromycin, oxacillin, kanamycin, lincomycin,
streptomycin, gentamicin, and chloramphenicol; 79% of the
strains are resistant to penicillin G. Of the clinical strains,
83% are resistant to penicillin G. All strains except strain
5717-1 are susceptible to the same antibiotics as the isolates
from healthy controls. Strain 5717-1 shows resistance to
penicillin G , lincomycin, and chloramphenicol.
Iso-c,,,,
Iso-Cl4,,
c14:0
Iso-c,,,,
Anteiso-C,,:,
Iso-Cl6:,
c16:0
ISO-C,,.,
Anteiso-C,,,,
Iso-Cl8~,
c18:0
Iso-c19:o
Anteiso-C,,,,
Iso-c*o:o
c*o:o
S . capitis
subsp.
ureolyficus
(n = 11)"
S . capitis
(n = 11)
S . caprae
(n = 5 )
S . epidermidis
( n = 31)
S . warneri
(n = 3)
1.91
5.14
1.79
11.08
25.34
1.26
5.52
3.36
2.70
0.44
12.03
2.58
0.68
0.27
25.83
1.67
4.87
0.73
7.42
27.37
0.70
2.66
3.05
3.41
0.04
11.66
3.00
0.89
0
32.83
2.04
3.00
2.70
19.23
20.63
0.75
5.88
4.31
1.73
0
14.89
2.85
0
0
21.39
0.95
3.82
1.13
9.66
30.16
0.90
2.76
2.95
3.39
0.53
10.61
3.33
1.75
0.22
28.10
0.52
4.58
6.35
7.90
27.37
1.41
4.34
1.72
2.44
0.40
10.50
1.21
0.94
0.50
29.58
n , Number of strains analyzed.
Cell wall peptidoglycan. The cell wall peptidoglycan type is
similar to that of S . cupitis and S . epidermidis on the basis of
the molar ratio of amino acids and is of the ~-Lys-Gly,.,Ser1.4 type.
The type strain of S . cupitis subsp. ureolyticus is strain
ATCC 49326 (= MAW 8436).
Description of the type strain. The type strain has all of the
uniform characteristics of the subspecies. In addition, it has
the properties described below. Cells are spherical (diameter, 0.8 to 1.0 p,m) and occur predominately in singles, pairs,
and clusters.
Agar colonies are circular, raised, entire, 5.5 mm in
diameter (after 5 days), smooth with glossy surfaces, and
yellow pigmented.
Nitrates are reduced to nitrites. Acetylmethylcarbinol is
produced. Weak pyrrolidonyl arylamidase is produced.
Weak hemolysins are detected.
Acid is produced aerobically from D-mannitol, a-lactose,
maltose, and sucrose. Weak acid is produced aerobically
from D-turanose. No acid is produced aerobically from
D-melezitose.
Resistant to penicillin G. There is variability within the
strain with respect to resistance to tetracycline, lincomycin,
and erythromycin; this variability is related to the presence
or absence of specific plasmids.
The guanine-plus-cytosine content of the DNA is 38.8
mol%.
Distinguishing characteristics. According to the Internutional Code of Nomenclature of Bacteria (13), S . capitis as
described by Kloos and Schleifer (9) should be designated S .
cupitis subsp. cupitis. The type strain is strain ATCC
27840T. S. cupitis subsp. ureolyticus can be distinguished
from S . capitis subsp. cupitis primarily by its positive urease
activity, its ability to produce acid aerobically from maltose,
its fatty acid profile, its larger colony size, and its DNA
relationship with S . cupitis subsp. capitis. The major features useful in distinguishing S . cupitis subsp. ureolyticus
from the closely related members of the S . epidermidis
species group (2, 4, 9, 17) are summarized in Table 4.
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VOL. 41, 1991
S . CAPITIS SUBSP. UREOLYTICUS SUBSP. NOV.
147
TABLE 4. Characteristics useful for differentiating S . capitis subsp. ureolyticus from other members of
the S. epidermidis species groupf’
Charac t e n st ic
S . capitis subsp.
ureolyticus
s. capitis
s. caprae
s. epider-
s. haem#-
midis
lyticus
S.hominis
S . warneri
*’
saccharolyticus
Colony size of 2 6 mm
Colony pigment
Anaerobic growth
Aerobic growth
Hemol ysis
Alkaline phosphatase activity
Urease activity
P-Glucosidase activity
P-Glucuronidase activity
Acid produced aerobically from:
D-Trehalose
D-Mannitol
D-Mannose
Maltose
Sucrose
Data from reference 6.
’ +, 90% or more of the strains are positive; -, less than 10% of the strains are positive; d , 11t o 89% of the strains are positive; ?,90% or more strains weakly
positive; ND, not determined. Parentheses indicate that the reaction is delayed.
ACKNOWLEDGMENTS
We thank A. G. Steigerwalt (Centers for Disease Control, Atlanta, Ga.) for determining the guanine-plus-cytosine content of
DNA, L. H. and N. R. Ericsson (AAA Laboratory, Mercer Island,
Wash.) for determining the amino acid content of the cell wall
peptidoglycan, M. Sasser (Microbial ID, Inc.) for determining the
cellular fatty acids, and C. G. George for the preparation of cell wall
peptidoglycans. We are also grateful to Jane Grant for typing the
manuscript.
This work was supported by Public Health Service grant 1ROI A1
21312 from the National Institute of Allergy and Infectious Diseases.
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