INTERNATIONAL
JOURNAL
OF SYSTEMATIC
BACTERIOLOGY,
Jan. 1975, p. 62-79
Copyright 0 1975 International Association of Microbiological Societies
Vol. 25, No. 1
Printed in U.S.A.
Isolation and Characterization of Staphylococci
from Human Skin
11. Descriptions of Four New Species: Staphylococcus warneri,
Staphylococcus capitis, Staphylococcus horninis, and Staphylococcus
s i m ul ans
WESLEY E. KLOOS AND KARL H. SCHLEIFER
Department of Genetics, North Carolina State University, Raleigh, North Carolina 27607, and Lehrstuhl f u r
Mikrobiologie, Universittit Mlinchen, 8 Munich 19, Germany
Staphylococci were isolated from the skins of people living in North Carolina
and New Jersey and were studied in an attempt to resolve their natural
relationships. As a result of this study, four new species are proposed in this
paper: Staphylococcus warneri, S. capitis, S . hominis, and S . simulans. The type
strains of these species are ATCC 27836, ATCC 27840, ATCC 27844, and ATCC
27848, respectively. The new species were established on the basis of a variety of
morphological, physiological, biochemical, and antibiotic characters. Cell wall
composition was particularly useful in resolving species and correlated well with
other characters. Characteristic pigment production was useful in distinguishing
several of the different species. A summary of the character variation found in the
species and a scheme for the classification of human cutaneous staphylococci are
included in this paper. The predominant staphylococci found on human skin
were S. epidermidis and S. horninis.
A brief account of the classification of staphylococci and their occurrence on human skin is
presented in the accompanying paper (15).Most
of the recent attempts a t classifying cutaneous
staphylococci have utilized the Baird-Parker
scheme (2-4, 10, 11). This scheme involves the
use of a small number of readily definable key
characters to identify isolates at the species and
subgroup levels. However, because of the small
number of characters used and the general lack
of information available on species character
variation, the scheme is limited in accuracy and
to the number of species and/or subspecies that
it can resolve.
The specific purpose of our investigation was
to identify the species of cutaneous staphylococci of humans. T o attempt this, we reevaluated existing taxonomic criteria, explored the
use of additional characters, and estimated
species character variation in cutaneous populations.
The results of studies on cutaneous strains of
Staphylococcus epidermidis (Winslow and
Winslow) Evans 1916, S. saprophyticus Fairbrother 1940, and three new species, s. cohnii,
S. haemolyticus, and S. xylosus, are presented
in the accompanying paper (15). The present
' Paper no. 4401 of the Journal Series of the North Carolina
Agricultural Experiment Station, Raleigh, N.C. 27607.
paper is concerned with the classification of four
additional new species of human cutaneous
staphylococci; it also contains summary data on
species character variation, occurrence of species on human skin, and a classification scheme.
MATERIALS AND METHODS
Bacterial strains. Staphylococci were isolated
from the healthy skins of two groups of people. One
group was composed of 20 people living in Raleigh,
N.C., who were sampled once each month for 6 to 13
months. The second group was composed of 20 people
living in New Jersey, who were sampled once during
the winter. Samples were taken from two separate
sites on the forehead and one site from one cheek, one
anterior and external nare, chin, each axilla, each
upper and lower arm, and each upper and lower leg.
Randomly selected representative strains of the
new species proposed in this paper are listed in Tables
1, 2, 3, and 4, respectively. (Additional strains that
were analyzed for only certain of the characters listed
in the tables are too numerous to be cited here, but
character information on these strains can be made
available upon request to the authors.)
Procedures for isolating staphylococci. Sampling
techniques and the composition of the isolation medium have been described previously (6).
Culture conditions. Culture conditions were similar to those previously described for the study of
cutaneous micrococci (6).
Character determinations. Procedures for determining deoxyribonucleic acid (DNA) base composi-
62
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VOL.25, 1975
STAPHYLOCOCCI
tion, colony morphology and pigment, cell morphology, motility, aerobic and anaerobic growth in thioglycolate, salt tolerance, growth temperature, catalase and benzidine activities, acetylmethylcarbinol
production, nitrate reduction, carbohydrate reactions, and susceptibility to various antibiotics, lysozyme, and lysostaphin were similar to those described
previously for micrococci (6). T h e minimal inhibitory
concentrations (MIC) of antibiotics and lysostaphin
are as denoted throughout the text.
Coagulase, hemolysis, deoxyribonuclease (DNase),
phosphatase, and bacteriolytic activities were determined by procedures described in the companion
paper (15).
Procedures for making cell wall hydrolysates and
determining peptidoglycan type, teichoic acid, configuration of lactic acid, and the anaerobic fermentation
of glucose have also been described previously (16).
RESULTS AND DISCUSSION
Characterization of Staphylococcus
species. The characteristics of the S. aureus
and other coagulase-positive strains isolated
from human skin in this study were similar to
those reported previously for various strains of
these organisms ( 3 , 14, 16).Descriptions of four
new Staphylococcus species isolated from
human skin are as follows.
(i) S. warneri sp. nov. (war. ner’i. M.L. gen. n.
warneri of Warner; named for Arthur Warner,
Jr., from whom this organism was originally
isolated.) Several strains tentatively identified
as members of this species were previously
designated S. epidernidis (CCM 2445; reference 13) or s. pyogenes albus (ATCC 155;
reference 16). Several strains, including SCH 5,
SCH 7, and SCH 13, were isolated by one of us
(K.H.S.) from dust and, based on their cell wall
composition and the configuration of the lactic
acid they produced, were placed together with
the above-mentioned strains in Staphylococcus
group I1 A3 (16). The following description of S.
warneri is based on a total of 38 strains, unless
noted otherwise.
Cells were gram-positive cocci, 0.5 to 1.2 pm
in diameter, nonmotile and nonsporeforming,
occurring predominantly in pairs and singly,
occasionally in tetrads. Only 12% of the strains
studied had approximately equal numbers of
pairs and tetrads.
Colonies on a P agar medium (6) were raised,
usually had a slightly elevated center, and were
circular, entire, smooth, glistening, and opaque.
They were small and had a diameter of 3 to 5
mm. The consistency of colonies or culture
streaks was usually sticky. Eighty percent of
the strains were pigmented to some extent.
Pigment occurred as a characteristic bright
yellow-orange or yellow ring around the edge of
FROM HUMAN SKIN. 11.
63
colonies or was a slight yellowish tint in the
center of colonies. Twenty percent of the strains
were unpigmented and had gray-white colonies.
Growth occurred in both the aerobic and
anaerobic portions of the thioglycolate medium,
indicating a facultatively anaerobic capability.
Growth in the anaerobic portion was usually
uniformly dense; however, 5% of the strains
demonstrated less growth, and 8% had only
individual colonies in the deeper, more anaerobic portion of this medium. Fifteen selected
strains demonstrated fermentation of glucose
by lowering the pH of a yeast extract-glucose
broth from 6.8 to 4.7-5.0 after anaerobic incubation. These strains produced nearly equal
amounts of D- and L-lactic acid from glucose.
All strains grew well at NaCl concentrations
up to 10%. Sixty-two percent of the strains grew
poorly or failed to grow at a NaCl concentration
of 15%. The optimal growth temperature range
was 25 to 40 C. Eighty-four percent of the
strains grew poorly or failed to grow at 15 C, but
92% grew well at 45 C.
All strains had either weak or moderate
catalase activity and were positive for the
benzidine test. All failed to produce coagulases.
Ninety-eight percent of the strains demonstrated weak or no hemolysin activity, 95%
demonstrated weak or no DNase activity, 74%
failed to reduce nitrates, and 78% failed to
demonstrate phosphatase activity; there was
variable bacteriolytic activity. All produced
acetylmethylcarbinol.
All strains produced acid aerobically from
glucose, fructose, sucrose, trehalose, and glycerol. Seventy-eight percent of the strains produced acid from maltose slowly, and 63% produced acid from mannitol. Fifty percent of the
strains produced acid from ribose, and 24%
produced acid from galactose. Only 2% of the
strains produced acid from mannose, and 11%
produced acid from lactose or turanose. All
failed to produce acid from rhamnose, xylose,
arabinose, gentiobiose, cellobiose, melezitose,
xylitol, sorbitol, inositol, salicin, adonitol, dulcitol, arabitol, erythritol, erythrose, raffinose,
melibiose, fucose, tagatose, lyxose, or sorbose.
All strains were resistant to lysozyme and
were slightly resistant to lysostaphin (MIC, 200
pg/ml). All were susceptible to erythromycin
(MIC, 0.4 to 1.6 pg/ml), tetracycline (MIC, 0.4
to 0.8 pg/ml), and novobiocin (MIC, 0.025 to 0.1
pg/ml). Seventy-eight percent of the strains
were susceptible to penicillin G (MIC, 0.012 to
0.025 pg/ml), and 98% were susceptible or
slightly resistant to streptomycin (MIC, 6.2 to
25 ,ug/ml).
Fifteen selected strains studied contained
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64
INT. J. SYST.BACTERIOL.
KLOOS AND SCHLEIFER
primarily on the basis of a combipeptidoglycan of the type L - L Y s - G ~ Y ~L. ~ - staphylococci
~.~,
Sero.6-l.4.
Their cell wall teichoic acid contained nation of its colony morphology, carbohydrate
reaction pattern, cell wall composition, lactic
glycerol and glucose.
The guanine plus cytosine (G+C) content of acid configuration, and the lack of nitrate
the DNA, as determined in six strains, was 34.7 reduction. There are also some significant differences between S . warneri and certain other
0.2 mol%.
Some variable characters and the G + C con- species in the parameters of pigment pattern,
tent of the DNA of some representative strains cell arrangement, anaerobic growth pattern in
thioglycolate, growth a t different NaCl concenof S. warneri are given in Table 1.
Strain ATCC 27836 (originally designated trations and temperature extremes, coagulase
AW 25) is the type strain of S. warneri. A activity, hemolysis, acetylmethylcarbinol production, and phosphatase, DNase, and bacteridescription of this strain follows.
Cells: Spheres, 0.8 to 1.0 pm in diameter, olytic activities. Several properties of's. warneri
occurring ill pairs, tetrads, and singly. Non- overlap with those of S. huemolyticus, S. epidermidis, and S. horninis, suggesting a relamotile and nonsporeforming. Gram positive.
Agar colonies: Circular, entire, 3.0 to 3.5 mm tively close relationship between these species.
(ii) S. cupitis sp. nov. (ca'pi. tis. L. noun cain diameter, raised with slightly elevated tenter, smooth with glistening surface, sticky con- p u t head; L. gen. noun capitis of the head; persistency. Originally opaque gray-white with yel- taining to that part of the human body where
low-orange edge; subcultures have lost pigment cutaneous populations of this species are usually the largest and most frequent.) Gordon (5)
and are now gray-white.
in 1905 reported a n unusual Staphylococcus
Catalase and benzidine tests are positive.
Chemoorganotroph; metabolism is respira- that he considered to be different from the
commonly recognized Staphylococcus epidertory.
midis albus. Strains of this unusual type were
Facultatively anaerobic.
Temperature relationships: Good growth a t different from those of the above-mentioned
species in that they failed to produce acid from
45 C, poor growth at 15 C.
Growth on NaCl agar: Good growth with 10% maltose or lactose but produced acid from
mannitol. They also were predominately found
NaC1, no growth with 15%.
on the scalp as compared to the hands, face, or
Coagulases not produced.
forearm. Although the few characters studied
Phosphatase not produced.
by Gordon will not conclusively identify his
Acetylmethylcarbinol produced.
isolate as belonging to S. capitis, nevertheless,
Nitrates not reduced.
Hemolysins: Weak hemolysis of bovine, they are consistent with those of S . capitis and,
hence, there is a reasonably good possibility
sheep, and human blood.
that he was observing this species. A later study
DNase activity is weak.
Acid produced aerobically from glucose, fruc- by Marples et al. (9) on the aerobic microflora of
tose, ribose, maltose, sucrose, trehalose, man- the scalp determined that the normal scalp
nitoll, and glycerol. No acid from galactose, cocci are predominately micrococci of type 3.
mannose, rhamnose, xylose, arabinose, lactose, However, according to Baird-Parker's scheme
gentiobiose, cellobiose, melezitose, xylitol, sop- (3), the characteristics of type 3 micrococci are
bitol, inositol, salicin, adonitol, dulcitol, arabi- not those of S. capitis. Hence, there is some
tol, erythritol, erythrose, raffinose, melibiose, uncertainty concerning earlier reports of possifucose, tagatose, lyxose, or sorbose.
ble encounters with this species. The following
Antibiotic susceptibilities: Slightly resistant description of S. capitis is based on a total of 47
to lysostaphin (MIC, 200 pg/ml) and strep- strains, unless noted otherwise.
tomycin (MIC, 12.5 pg/ml). Susceptible to peniCells were gram-positive cocci, 0.8 to 1.2 pm
cillin G (MIC, 0.025 pg/ml), erythromycin in diameter, nonmotile and nonsporeforming,
(MIC, 1.6 pg/ml), tetracycline (MIC, 0.8 pg/ occurring predominantly in pairs and tetrads.
ml), and novobiocin (MIC, 0.025 pg/ml). ResistColonies on a P agar medium (6) were slightly
ant to lysozyme.
convex, circular, entire, smooth, slightly dull to
Cell wall peptidoglycan: L-LYs-G~Y,.,, L- glistening, opaque and usually a distinctive
chalk white (unpigmented). They were very
Ser,.,.
Cell wall teichoic acid composition: Glycerol small and had a diameter of 1 to 3 mm.
Growth occurred in both the aerobic and
and glucose.
anaerobic portions of the thioglycolate medium,
G - t C content of the DNA: 34.2 mol%.
S. warneri can be distinguished from all other indicating. a facult ativelv anaerobic caDa b ilitv.
-
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VOL.25, 1975
STAPHYLOCOCCI FROM HUMAN SKIN. 11.
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66
KLOOS AND SCHLEIFER
INT.J. SYST.BACTERIOL.
Growth in the anaerobic portion was usually of teichoic acid, and cell wall hydrolysates had
uniforirnly dense; however, 11% of the strains to be concentrated for the detection of free
demonstrated less growth, and 21% had only glycerol. Cell walls also contained additional
individual colonies in the deeper, more anaero- glutamic acid. In partial acid hydrolysates,
bic portion of this medium. Nineteen selected diglutamic acid was found, indicating that a
strains of this species demonstrated the fermen- glutamic acid polymer is present. The additation of glucose by lowering the pH of a yeast tional glutamic acid can be extracted with hot
extract-glucose broth from 6.8 to 4.2-4.7 after trichloroacetic acid like the teichoic acid.
anaerobic incubation. These strains also proThe G+C content of the DNA, as determined
duced predominately L-lactic acid and between in five strains, was 33.6 + 1.0 mol%.
5 and 25% D-lactic acid from glucose.
Some variable characters and the G+C conNinety percent of the strains grew well a t tent of the DNA of some representative strains
NaCl concentrations up to 10%. All strains of S. capitis are given in Table 2.
either grew poorly or failed to grow a t a NaCl
Strain ATCC 27840 (originally designated LK
concentration of 15%.The optimal growth tem- 499) is the type strain of S. cupitis. A descripperature range was 30 to 40 C. All strains failed tion ofthis strain follows.
Cells: Spheres, 0.9 to 1.2 p m in diameter,
to grow a t 15 C but grew well a t 45 C.
All strains had either weak or moderate occurring predominantly in tetrads and pairs.
catalase activity and were positive for the Nonmotile and nonsporeforming. Gram posibenzidine test. All failed to produce coagulases. tive.
Agar colonies: Circular, entire, 2 to 2.5 mm in
All strains demonstrated either weak or no
hemolysin activity on bovine or sheep blood but diameter, slightly convex, smooth with glistenusually produced good hemoly sin activity on ing surface. Opaque, chalk white.
Catalase and benzidine tests are positive.
human blood. Strains had variable acetylmethC hemoorganotroph; metabolism is respiraylcarbinol production and had weak or no
phosphatase activity. Eighty-three percent of tory.
the strains reduced nitrates, and all strains
Facultatively anaerobic.
demonstrated weak DNase and moderate to
Growth a t extreme temperatures: Good
strong b acteriolytic activities.
growth at 45 C, no growth at 15 C.
Growth on NaCl agar: Good growth with 10%
By comparison with other staphylococci, S .
cupitis produced acid aerobically from only a NaC1, poor growth with 15%.
Coagulases not produced.
few different carbohydrates. All strains proPhosphatase not produced.
duced acid from glucose, fructose, mannose,
Acetylmethylcarbinol not produced.
and ghrcerol. Ninety-one percent of the strains
produced acid slowly from sucrose, and 94%
Nitrates are reduced.
Hemolysins: No hemolysis of bovine and
produced acid slowly from mannitol. Only 2%of
the strains produced acid from trehalose or sheep blood, but moderate hemolysis of human
lactose. All failed to produce acid from galac- blood.
tose, rhamnose, xylose, arabinose, ribose, malDNase activity is weak.
tose, turanose, gentiobiose, cellobiose, meleziAcid produced aerobically from glucose, fructose, xylitol, sorbitol, arabitol, inositol, salicin, tose, mannose, sucrose, mannitol, and glycerol.
adonitol, dulcitol, erythritol, erythrose, raf- No acid from galactose, rhamnose, xylose, arabfinose, melibiose, fucose, tagatose, lyxose, or inose, ribose, maltose, lactose, trehalose, turansorbose.
ose, gentiobiose, cellobiose, melezitose, xylitol,
All strains were resistant to lysozyme and sorbitol, inositol, salicin, adonitol, dulcitol,
were slightly resistant to lysostaphin (MIC, 200 arabitol, erythritol, erythrose, raffinose, melipg/ml). All were susceptible to erythromycin biose, fucose, tagatose, lyxose, or sorbose.
Antibiotic susceptibilities: Slightly resistant
(MIC, 0.4to 0.8 pg/ml), streptomycin (MIC, 1.6
to 6.2 pg/ml), and novobiocin (MIC, 0.05 to 0.1 to lysostaphin (MIC, 200 pg/ml); susceptible to
pg/ml). Ninety-four percent of the strains were penicillin G (MIC, 0.05 pg/ml) , erythromycin
susceptible to penicillin G (MIC, 0.025 to 0.05 (MIC, 0.4 pg/ml), streptomycin (MIC, 3.1 pg/
pglml), and 85% were susceptible to tetracy- ml), tetracycline (MIC, 0.4 pg/ml), and novobicline (MIC, 0.4 to 0.8 pg/ml).
ocin (MIC, 0.05 pg/ml); resistant to lysozyme.
Nineteen selected strains contained peptidoCell wall peptidoglycan: ~ - L y s - G l y1,~ . Lglycan of the type ~ - L y s - G l y ~ . L-Sero.e-1.2.
~-~.~,
Ser,.o.
Cell wall teichoic acid: Glycerol.
Their cell wall teichoic acid contained glycerol,
G+C content of the DNA: 35.5 mol%.
and no neutral sugar or additional amino sugar
was detected. Cell walls contained low amounts
S. cupitis can be distinguished from all other
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ND
ND
ND
ND
ND
ND
ND
ND
35.8
NDd
ND
ND
ND
ND
ND
ND
35.5
31.4
31.0
34.1
+
+
*C
+
*C
+
t
*C
+
*C
+
+
t
+
+
*
+
+
+
*
4.7
4.3
4.7
4.3
4.5
4.3
4.7
4.6
4.4
ND
I
4.5
4.4
4.6
4.2
4.6
4.6
4.5
4.6
4.4
4.7
Anaerobic
fermentation of glucose (pH)
91
92
82
95
90
87
88
ND
90
92
90
91
90
81
89
92
90
75
87
89
L-Lact i c
acid (%)
-
f
*
*
+
+
-
f
+
f
-
*
-
f
-
-
f
-
+
+
t
-
42
+
+
+
t
+
+
+
+
+
+
+
+
+
+
+
-
*
f
-
-
-
-
-
+
+
+
+
+
+
+
+
++
+
+
+
+
+
+
+
+
+
++
++
+
+
+
f
+
i
+
-
-
I
_
++
+
+
+
-
+
++
++
++
+
+
+
+
+
+
+
+
+
t+
+
_
.
crose nose iitol
s u - vIan I a n
-
0.050
0.050
50.0
0.025
0.050
0.050
0.050
0.050
0.050
0.050
0.200
0.025
0.050
0.050
0.050
0.050
0.050
0.050
0.050
50.0
Acid (aerobically)
from
Penicillin
G MIC
(/Lg/mU
/___
I
Growth
Acetyl- Nitrate
PhosHemolon 15%
methyl- reducp ha t ase
ysis
tion
carbinol
NaCl agar
0.4
50.0
50 .O
0.4
0.4
0.8
0.4
0.8
0.4
0.8
50.0
0.4
0.4
0.4
0.8
0.4
0.4
0.8
0.4
50.0
Tetracyline
MIC
(rdml)
* Moles of glutamic acid
1.0
1.1
1.1
1.0
1.1
3.8
3.9
4.3
3.9
3.8
ND ND
4.1
3.5
4.0
4.1
3.7
3.9
3.5
3.8
4.0
3.8
1.7
1.6
1.7
1.4
1.a
1.6
1.4
1.5
1.6
1.0
1.0
1.0
1.2
1.0
1.0
0.8
0.9
1.0
1.1
Ser- Clyine cine
ND
1.6
1.4
1.5
1.8
1.4
1.6
1.3
1.5
1.6
1.6
Cell wall
glutamic acid
(mol/mol
lysine)*
Symbols for anaerobic growth: +, uniform dense; i,gradient of growth from dense to light down the tube; *C, gradient of growth plus individual colonies.
per mole of lysine is 1.0 in all other staphylococci.
Reaction: ++, strong positive (carbohydrates only); +, positive; f, weak; -, negative.
ND, Not determined.
ATCC 27840
ATCC 27841
ATCC 27842
ATCC 27843
RM 390
GS 14
KES 14
SE 21
CK 17
RK 23
KH 220
PM 378
WK 11
WK 445
DBM 213
E K 11
HM 11
BS 2
CE 2
S K 13
Strain
Mol 70
G+C
Anaerobic
thioglycolate
growth@
TABLE
2 . Variable characters of 20 strains of Staphylococcus capitis
68
INT. J. SYST.BACTERIOL.
KLOOS AND SCHLEIFER
staphylococci primarily on the basis of its small
and slightly convex, chalk-white colony, carbohydrate reaction pattern, cell wall composition,
and lactic acid configuration. There are also
some significant differences between S. cupitis
and certain other species in the parameters of
cell arrangement, anaerobic growth pattern in
thioglycolate, growth a t different NaCl concentrations and temperature extremes, coagulase
activity, hemolysis, acetylmethylcarbinol production, nitrate reduction, and phosphatase,
DNase, and bacteriolytic activities.
(iii) S. horninis sp. nov. (ho’mi.nis. L. noun
homo man; L. gen. noun hominis of man;
named for the host on whose skin this species is
commonly found.) The following description of
S. horninis is based on a total of 240 strains,
unless noted otherwise.
Cells were gram-positive cocci, 1.0 to 1.5 pm
in diameter, nonmotile and nonsporeforming,
occurring predominantly in tetrads with occasional pairs. Thirty-eight percent of the strains
had approximately equal numbers of tetrads
and pairs.
Colonies on a P agar medium ( 6 ) were raised
to very slightly umbonate and, with age, usually
developed a wide beveled edge and elevated
center. The older colony profile appears to be
species specific. Colonies were small, circular,
and usually entire, smooth dull, and opaque.
Their diameter was 3 to 4 mm. Colony pigment
was variable; 64% of the strains had yelloworange or yellowish pigment in the center of
the colonies and 36% had gray-white (unpigmented) colonies. Older colonies ( 2 6 days)
often showed concentric rings of lighter and
darker colors.
Growth occurred in the aerobic portion and
84% of strains produced slight growth in the
anaerobic portion of the thioglycolate medium,
indicating a limited facultatively anaerobic
capability. Growth was much less intense in the
deeper, more anaerobic, portion of the medium
and was usually associated with the presence of
a few very small individual colonies. Sixteen
percent of the strains failed to produce any clear
signs of visible anaerobic growth. Eighteen
selected strains demonstrated the fermentation
of glucose by lowering the pH of a yeast extractglucose broth from 6.8 to 4.4-4.9 after anaerobic
incubation. Considering the relatively poor anaerobic growth of this species, it seemed somewhat unusual to obtain acid from glucose under
anaerobic conditions. However, it should be
mentioned here that this species rapidly produces very strong acid from glucose aerobically,
and even slight anaerobic growth or a slight
amount of oxygen present in the glucose medium might result in the production of significant quantities of acid such as were observed.
The above-mentioned 18 strains could be subdivided into two groups on the basis of the type of
lactic acid isomers produced from glucose. One
group contained eight strains that produced
only D-lactic acid, and another group contained
10 strains that produced both L- and D-laCtiC
acid. The latter strains may contain, in addition
to a D-laCtate dehydrogenase, an L-lactate dehydrogenase. Such differences were also noted in
S. aureus by Schleifer and Kocur (16). Most S.
uureus strains produce L- and D-lactic acid, but
a few strains produce only L-lactic acid. Ninetyfive percent of the strains grew well a t NaCl
concentrations up to 7.5%, but 97% grew poorly
a t 10% and failed to grow at 15%. The optimal
growth temperature range was 28 to 40 C.
Seventy-two percent of the strains failed to grow
a t 15 C, and all strains grew well a t 45 C.
All strains had either weak or moderate
catalase activity and were positive for the
benzidine test. All failed to produce coagulases
and had weak or no hemolysin or DNase activities. Eighty-three percent of the strains had no
phosphatase activity, weak or no bacteriolytic
activity, and reduced nitrates. Strains had
variable acetylmethylcarbinol production.
All strains produced acid aerobically from
glucose, fructose, maltose, sucrose, and glycerol. Ninety percent of the strains produced
acid from trehalose, 81% produced acid from
turanose, 60% produced acid from lactose, 52%
produced acid from galactose, and 51% produced acid from melezitose. Only 7% of the
strains produced acid from mannitol, and 10%
of the strains produced acid from mannose. All
failed to produce acid from rhamnose, xylose,
arabinose, ribose, gentiobiose, cellobiose,
xylitol, sorbitol, inositol, salicin, adonitol, dulcitol, arabitol, erythritol, erythrose, raffinose,
melibiose, fucose, tagatose, lyxose, or sorbose.
All strains were resistant to lysozyme and
were slightly resistant to lysostaphin (MIC, 100
to 200 pg/ml). Seventy-seven percent of the
strains were susceptible to penicillin G (MIC,
0.006 pglml), 97% were susceptible to streptomycin (MIC, 1.6 to 6.2 pg/ml), 93% were
susceptible to erythromycin (MIC, 0.4 to 0.8
pg/ml), 64% were susceptible to tetracycline
(MIC, 0.4 pg/ml), and 99% were susceptible to
novobiocin (MIC, 0.1 to 0.2 pg/ml). A relatively
large proportion of the strains (36%) was resistant to tetracycline.
Eighteen selected strains contained peptidoglycan of the type ~ - L y s - G l y ~ . ~
L-Sero.6-1.3.
-~.~,
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VOL.25, 1975
STAPHYLOCOCCI FROM HUMAN SKIN. 11.
69
Cell wall teichoic acid composition: Glycerol
Their cell wall teichoic acid contained glycerol
and, in addition, had 0.3 t o 0.8 mol of glucosa- and glucosamine.
mine per mol of glycerol. No neutral sugar was
G+C content of the DNA: 33.7 mol%.
S . hominis can be distinguished from all other
found. This species produced only low levels of
teichoic acids and, like S. capitis, required staphylococci primarily on the basis of a combiconcentration of the cell wall hydrolysates for nation of its colony morphology and pigment
the detection of free glycerol.
pattern, predominant tetrad cell arrangement,
The G + C content of the DNA, as determined poor or no detectable growth in thioglycolate,
in five strains, was 33.0 & 1.0 mol%.
low NaCl tolerance, and carbohydrate reaction
Some variable characters and the G+C con- pattern. There are also some significant differtent of the DNA of some representative strains ences between s. hominis and certain other
of S. hominis are given in Table 3.
species in the parameters of cell wall composiStrain ATCC 27844 (originally designated tion, lactic acid configuration, temperature exDM 122) is the type of strain of S. hominis. A tremes of growth, coagulase activity, hemolysis,
acetylmethylcarbinol production, nitrate reducdescription of this strain follows.
Cells: Spheres, 1.2 to 1.4 pm in diameter, tion, and phosphatase, DNase, and bacterioccurring predominantly in tetrads and occa- olytic activities. Several of these properties
sionally in pairs. Nonmotile and nonsporeform- overlap with those of S. epidermidis, S. h e moly ticus, and S. warneri, suggesting a close
ing. Gram positive.
Agar colonies: Circular, entire, 4 to 4.5 mm in relationship among these species.
(iv) S. sirnulans sp. nov. (sim’u,lans. L. part.
diameter, slightly umbonate with wide, beveled
edge and elevated center, smooth dull surface. adj. simulans imitating; named for having simiYellow-orange pigment is produced in the ten- larities to certain coagulase-positive staphylococci, including S. aureus.) The following deter of opaque colonies.
scription of S . simulans is based on a total of 13
Catalase and benzidine tests are positive.
C hemoorganotroph; metabolism is respira- strains.
Cells were gram-positive cocci, 0.8 to 1.5 pm
tory.
in diameter, nonmotile and nonsporeforming,
Slightly facultatively anaerobic.
Growth a t extreme temperatures: Good occurring predominantly in pairs and tetrads
and occasionally singly.
growth at 45 C, no growth at 15 C.
Colonies on a P agar medium (6) were slightly
Growth on NaCl agar: Good growth with 7.5%
NaC1, slight growth with lo%, no growth with raised, circular, entire, smooth, slightly glistening, translucent to slightly transparent, and
15%.
usually gray-white (unpigmented) . TwentyCoagulases not produced.
three percent of the strains had a very slight tint
Phosphatase not produced.
of yellowish pigment. The colonies were relaAcetylmethylcarbinol produced weakly.
tively large and had a diameter of 5.0 to 7.5 mm.
Nitrates are reduced.
Growth occurred in both the aerobic and
Hemolysins: No hemolysis of bovine, sheep,
anaerobic portions of the thioglycolate medium,
or human blood.
indicating a facultatively anaerobic capability.
DNase activity is weak.
Acid produced aerobically from glucose, fruc- Growth in the anaerobic portion of the medium
tose, galactose, maltose, lactose, sucrose, treha- was uniformly dense. This species demonlose, turanose, and glycerol. No acid from man- strated the fermentation of glucose by lowering
nose, rhamnose, xylose, arabinose, ribose, me- the pH of a yeast extract-glucose broth from 6.8
lezitose, mannitol, gentiobiose, cellobiose, to 4.7-5.0 after anaerobic incubation. The
xylitol, sorbitol, inositol, salicin, adonitol, dul- strains could be subdivided into two groups on
citol, arabitol, erythritol, erythrose, raffinose, the basis of the type of lactic acid isomers
produced from glucose. One group contained six
melibiose, fucose, tagatose, lyxose, or sorbose.
Antibiotic susceptibilities: Slightly resistant strains that produced only L-lactic acid, and
to lysostaphin (MIC, 100 pg/ml); susceptible to another group contained seven strains that
penicillin G (MIC, 0.006 pg/ml), streptomycin produced both L- and D-lactic acid. The occur(MIC, 6.2 pg/ml), erythromycin (MIC, 0.4 pg/ rence of strains producing L- and D-laCtiC acid or
ml), and novobiocin (MIC, 0.1 pg/ml); resistant only L-lactic acid was also found in S . aureus
to tetracycline (MIC, 100 pg/ml); resistant to (16).
All strains grew well a t NaCl concentrations
lysozyme.
up to 10%. Seventy-seven percent of the strains
1,
LCell wall peptidoglycan: L-LYS-G~Y,.
grew poorly at a NaCl concentration of 15%,and
Ser,.,.
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On: Mon, 31 Jul 2017 16:44:45
ND
ND
ND
35.8
ND
ND
ND
ND
ND
ND
ND
ND
ND
33.7
32.3
NDd
29.9
ND
ND
33.5
f
Y
!
f
fC
-
f
-
Y
-
2
f
fC
YO
Y
Y
Y
-C
-
fC
fC
fC
-C
-C
f
YO
YO
Y
-
-
-
-
YO
YO
4.5
4.5
4.6
4.9
4.5
4.4
4.6
4.6
4.5
4.4
4.5
4.5
4.4
4.5
4.5
4.5
4.5
4.6
ND
ND
0
0
0
50
0
43
38
0
49
49
0
0
42
51
0
37
47
45
ND
ND
+
f
f
f
f
f
+
-
-
-
f
-
f
-
+
f
f
-
+
+
+
+
f
-
-
+
+
f
+
+
+
f
+
+
+
+
+
-
-
-
s
8
2
aJ
-
+
+
+
-
+
+
+
+
-
-
+
+
+
+
+
+
+
+
+
+
+
__ -
z
x
m
-%
W
++ ++
f
++
f
+
++ +
++ +
++ ++
++ ++
++ ++
++ ++
++
++ ++
++ + +
++
f
+
+ ++
++ ++
++ f
++
++ ++
-
-
3
+-'
0
aJ
-
Acid (aerobically) from
0.006
0.006
0.006
6.2
0.006
12.5
0.006
25.0
6.2
0.006
0.050
0.050
0.006
0.006
0.006
12.5
0.006
6.2
0.006
0.006
0.4
0.4
0.4
0.4
0.4
0.4
0.8
400.0
0.4
0.4
0.4
0.8
8.4
0.8
200.0
0.8
400.0
200.0
0.4
0.4
100.0
100.0
0.4
50.0
0.4
0.4
0.4
100.0
200.0
0.4
0.4
0.4
100.0
0.4
100.0
200.0
0.4
100.0
0.4
0.4
u
h
.-E
ND
ND
4.1
4.3
3.9
3.8
3.9
3.6
3.8
4.0
3.6
4.1
3.8
4.4
4.1
4.3
3.3
3.5
3.6
3.9
- -
0.8
0.6
1.o
1.1
0.9
1.3
1.2
1.o
1.3
0.9
1.2
0.7
0.8
0.9
1.1
1.3
1.1
0.6
ND
ND
mE 5
- -
.d
0)
a
0.5
0.5
0.3
0.4
0.8
ND
0.5
0.7
0.5
0.8
0.5
0.3
0.4
0.4
0.5
0.4
0.8
0.3
ND
ND
Peptidoglycan
:mol/mo
of glu- Teichoic
tamic acid (mol
Peni- Erythro. Tetra:illin G mycin cycline
acid)
of glucoMIC - - saminel
MIC
MIC
mol of
rglml) (rglml) (rglml)
glycerol)
Symbols for colony pigment: YO, yellow-orange center; Y, yellowish center; -, gray-white (unpigmented).
Symbols for anaerobic growth: f, gradient of growth from moderate to light down the tube; +C, gradient of growth plus individual colonies; -C, individual colonies only
(usually <50); -, no detectable growth.
Reaction: ++, strong positive (carbohydrates only); +, positive; f ,weak; -, negative.
ND. Not determined.
ATCC 27844
ATCC 27845
ATCC 27846
ATCC 27847
WK52
PM 375
S M 67
J L 248
T W 52
T W 139
GH 149
KH 211
AW 178
RM 122
MCS 13
GS 16
DGS 13
BS 14
LK 47
MK 314
Strain
BacAnaerobic
LInaerobic
Niferments
Zolony
He- AcetylDeoxy- teriothioglyMol ?6
Lactic
trate
of
moly- met hy 1ribonu- lytic
Pigmeduc.
colate
acid
G + C menta
:arbinol
sis
clease activtion
(%)
ity
TABLE
3 . Variable characters of 20 strains of Staphylococcus hominis
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1
ND
ND
37.2
4.7
4.7
4.8
4.8
4.8
4.8
4.9
4.8
4.9
4.8
4.9
4.8
5.0
(PW
100
63
60
100
100
64
100
69
70
75
f
f
+
-
0
f
-
+
i
-a
NaCl
agar
:r:t
+, positive; +, weak;
'
I
-, negative.
l l
I
*
-
-
-
1 --
1 ' I
Acid (aerobically) from
-
+
+
-
+
~
+
~
++
++
+
*
+
+
+ + *
++
++
+ + - ++ +
+
++ ++
+ - - + + +
++ - +
+
f
++
- * * + + *
+ + - + ++
Galac- Man- Ri- Malt- Tretose
nose bose ose halosf
-
Growth Acetyl- Nitrate Phos- Bacterimethyl- reduc- phaolytic
at l5
carbinol
tion
tase activity
Reaction: ++, strong positive (carbohydrates only);
ND, Not determined.
AW232
SM 128
SM 81
KL 299
KL 104
MAW 222
HK 12
D W 196
CE 13
ATCC 27848
ATCC 27849
ATCC 27850
ATCC 27851
Strain
Anaerobic
fermenta- L-Lactic
Mol
tion of
acid
G + C glucose
1'(
I
TABLE
4. Variable characters of 13 strains of Staphylococcus simulans
1
3.1
0.012
0.025
1.6
0.012
0.025
25.0
0.012
0.012
0.025
25.0
12.5
0.800
Penicillin G
1
0.8
0.4
400.0
0.8
0.4
0.8
400.0
0.4
400.0
0.8
0.8
0.8
0.4
200.0
0.4
200.0
400.0
100.0
0.8
0.4
0.4
200.0
0.8
0.4
200.0
6.2
Erythro- Tetramycin cycline
MIC
MIC
(ccdml) (ccg/mU
I
0.2
0.1
0
0.1
0.2
0.1
0
0.1
0.2
0
0
0
0
4.9
5.7
6.0
4.7
5.6
5.3
5.1
5.0
4.8
5.2
4.8
5.4
5.0
Ser- Glyine cine
72
INT.J. SYST.BACTERIOL.
KLOOS AND SCHLEIFER
23% failed to grow a t this concentration. The
optimal growth temperature range was 25 to
40 C. Eighty-five percent of the strains grew
well a t 15 C, and all grew well a t 45 C.
All strains had weak or moderate catalase
activity and were positive for the benzidine test.
All failed to produce coagulases. All strains
demonstrated weak or no hemolysin activity on
bovine or sheep blood but produced weak to
good hemolysin activity on human blood. Seventy-seven percent of the strains failed to produce acetylmet hylcarbinol or had weak phos-
phatase activity. Ninety-two percent of the
strains reduced nitrates or demonstrated moderate to good bacteriolytic activity. All had
weak to moderate DNase activity.
All strains produced acid aerobically from
glucose, fructose, lactose, sucrose, and glycerol.
Seventy-seven percent of the strains produced
acid from mannose and trehalose, and 92%
produced acid from mannitol. Fifty-four percent of the strains produced acid from ribose.
Sixty-nine percent of the strains failed to produce acid from maltose; 31% produced only
TABLE5 . Morphological characters of Staphylococcus species
~
~
Colony pigment
T
Total
Colony
no. of
diametera
strains
(mm)
studied
Species
Colony
Colony profile
CI
S . aureus . . . . . . . . .
S . simulans . . . . . . .
s. xylosus
........
S . cohnii . . . . . . . . .
S . saprophyticus . .
S . haenolyticus . .
S . warneri . . . . . . . .
S . horninis . . . . . . . .
S. epidermidis . . . .
S . capitis . . . . . . . . .
,
46
13
102
42
83
117
38
240
204
47
7.1 0.1
6.5 0.2
7.0 0.1
5.9 i 0.2
6.5 0.1
5.4 0.1
3.8 mo.1
3.7 * 0.05
3.1 * 0.04
2.4 * 0.07
*
*
*
5
21
18
13
*
1
iand s %.
Percentages of strains possessing the character
12
21
11
32
20
36
8
53
29
23
100
100
71
7
20
13
17
10
80
87
22
18
96 I 1 0 0
100
100
68
95
98
92
98
91
99
100
12
83
78
82
1100
15
32
5
2
8
2
9
1
jicated.
TABLE
6. Physiological characters
of Staphylococcus species
Growth on:
Anaerobic growth in thioglycolate
semisolid medium
Species
S . aureus . . . . . . . . . . . . . . .
S . simulans . . . . . . . . . . . . .
s. xyl0.sus . . . . . . . . . . . . . .
S. cohnii . . . . . . . . . . . . . . .
S. saprophyticus . . . . . . . .
S . haemolyticus . . . . . . . .
S . warneri . . . . . . . . . . . . . .
S . horninis . . . . . . . . . . . . .
S . epidermidis . . . . . . . . . .
S . capitis . . . . . . . . . . . . . . .
Total no.
of strains
studied
46
13
102
42
83
117
38
240
204
47
Gradi- ScatUni- ent: tered
formly dense colodense
to
nies
light
> 50
986
100
68
24
59
20
87
98
68
Scattered
Noni
colonies
<50
-
2
11
47
30
8
5
10
1
11
14
12
38
17
70
61
2
14
14
17
9
66
8
44
1
21
OGrowth: +, good; *, weak; -, none.
Percentages of strains possessing the character indicated.
4
7
1
4
3
5
1
2
30
16
Growth at:
10% NaCl
15% NaCl
agar
agar
+
15 C
i
+
- -
-
---
96
100
100
100
100
100
100
40
77
48
64
70
45
54
3
4
51
60 60 36
4
23 85 15
14 100
15 100
100
40
43 57
8 76 16
38
97
28 72
21 79
96
LOO
49
+a
4
97
16 79
90 10
--
38
21
30
15
8
-
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-
-
+ * -
-
-
100
100
19 19 62
76 17 7
38 36 26
100
92 8
100
100
100
VOL.25, 1975
73
STAPHYLOCOCCI FROM HUMAN SKIN. 11.
weak acid from this carbohydrate. Eight percent of the strains produced weak acid from
galactose. All failed to produce acid from rhamnose, xylose, arabinose, turanose, gentiobiose,
cellobiose, melezitose, xylitol, sorbitol, inositol,
salicin, adonitol, dulcitol, arabitol, erythritol,
erythrose, raffinose, melibiose, fucose, tagatose,
lyxose, or sorbose.
All strains were resistant to lysozyme and
were susceptible to lysostaphin (MIC. 12.5 to 25
pg/ml). All were susceptible to streptomycin
(MIC, 1.6 to 3.1 pg/ml) and novobiocin (MIC,
0.1 to 0.2 pg/ml). Fifty-four percent of the
strains were susceptible to penicillin G (MIC,
0.012 pg/ml), and 77% were susceptible to
erythromycin (MIC. 0.4 to 0.8 pg/ml). Fortysix percent of the strains were susceptible to
tetracycline (MIC, 0.4 pg/ml). This species
contained peptidoglycan of the type L-LYSGly,-,. The cell wall teichoic acid contained
glycerol and galactosamine.
The G + C content of the DNA, as determined
in six strains, was 35.6 f 0.6 mol%.
Some variable characters and the G + C content of the DNA of some representative strains
of S . simulans are given in Table 4.
Strain ATCC 27848 (originally designated
MK 148) is the type strain of S . simulans. A
description of this strain follows.
Cells: Spheres, 0.8 to 1.0 pm in diameter,
occurring predominantly in pairs and tetrads
and occasionally singly. Nonmotile and nonsporeforming. Gram positive.
Agar colonies: Circular, entire, 6.5 to 7.0 mm
in diameter, slightly raised, smooth with
slightly glistening surface. Translucent to
slightly transparent and gray-white (unpigmented).
Catalase and benzidine tests are positive.
Chemoorganotroph; metabolism is respiratory.
Facultatively anaerobic.
TABLE
7. Biochemical characters of Staphylococcus species
I
Species
I
I
Total no. Coagulase
of strains
studied
S . cohnii . . . . . . . . . . . . . . .
S. saprophyticus . . . . . . . .
S . haemolyticus . . . . . . . .
S . warneri . . . . . . . . . . . . . .
S . hominis . . . . . . . . . . . . .
S . epidermidis . . . . . . . . . .
S. c a p i t i s . . . . . . . . . . . . . . .
46
13
102
42
83
117
38
240
204
47
1 1
methylNitrate
~~~~~1
reduction
production
I
I
Phosphatase
+
-
+a
s. aureus . . . . . . . . . . . . . . .
S.simulans . . . . . . . . . . . . .
s. xylosus . . . . . . . . . . . . . .
Hemolysis
+
100b
Deoxyribonuclease
g
Bacteriolytic
activity
100
100
100
100
100
100
100
10 12
<1 16 83
89 6
100
100
100
100
“Reactions: +, positive; +, weak; -, negative.
’Percentages of strains giving the reaction indicated.
TABLE
8. Carbohydrate reactions
of Staphylococcus species: monosaccharides under aerobic conditions
D(+)
Species
S.aureus . . . . . . . . . . . . . .
S . simulans . . . . . . . . . . . .
s. rylosus . . . . . . . . . . . . . .
S . cohnii . . . . . . . . . . . . . . .
S . saprophyticus . . . . . . . .
S . haemolyticus . . . . . . . .
S. warneri . . . . . . . . . . . . . .
S. hominis . . . . . . . . . . . . .
S . epidermidis . . . . . . . . . .
S. c a p i t i s . . . . . . . . . . . . . . .
a
Reactions:
Total no.
of strains
studied
46
13
102
42
83
117
38
240
204
47
Glucose
1
B-D(-)
Fructose
1
I
D(+)
D(+)
Galactose
Mannose
-
1
I
D(+)
Xylose krabinose
L(+)
7
++=+
++ +
-
++,
100
100
100
8 92
100
62 29 9
56 38 6 95 5
10 90
86 12 2 100
8 92
85 14 1 92 5 3
24 36 40 29 24 47
98 2
100
5 19 76
79 21
100
33 19 48
100
80 5 15
100
100
79 21
100
100
+ +, strong acid; +, moderate acid;
*, weak acid;
Ribose
D(-)
++,
~
1OOb
’Percentages of strains giving reaction indicated.
I
+
89
77
95
51
3
2
- , negative.
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100
100
100
100
100
74
INT. J. SYST.BACTERIOL.
KLOOS AND SCHLEIFER
I
I 1
I
I
I
r -w0
+
I
I
*r3s*mzm*o*
CDmmcvmr-moTrtmm
.
..
.
...
.
..
.
.
..
.
.. ..
. .. ..
.
..
.
...
.
.
..
.
...
.
.
..
.
...
.
.
..
.
...
.
.
..
.
...
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Growth a t extreme temperature: Good
growth a t 15 and 45 C.
Growth on NaCl agar: Good growth with 10%
NaC1, no growth with 15%.
Coagulases not produced.
Phosphatase activity weak.
Acetylmethylcarbinol not produced.
Nitrates are reduced.
Hemolysins: No hemolysis of sheep blood,
Species
S . aureus . . . . . . . . . . .
S . simulans . . . . . . . . .
s. xylosus . . . . . . . . . .
S . cohnii.. . . . . . . . . . .
S . saprophyticus . . . .
S . haemolyticus . . . . .
S . warneri . . . . . . . . . .
S . hominis . . . . . . . . . .
S . epidermidis . . . . . .
S . capitis . . . . . . . . . . .
75
STAPHYLOCOCCI FROM HUMAN SKIN. II.
VOL.25, 1975
Total
Level of susceptibility"
no. of
strains 12.5 25 50 100 200
studied (+) (+) ( + I ( + I (*I
10
23
51
13
101
42
83
116
38
178
152
48
82
77
27
8
67
100
28
6
72
34
8
41
68
8
66
92
59
32
92
weak hemolysis of bovine blood, good hemolysis
of human blood.
DNase activity weak.
Acid produced aerobically from glucose, fructose, ribose, maltose (very weak), lactose, sucrose, trehalose, mannitol, and glycerol. No acid
from galactose, mannose, rhamnose, xylose,
arabinose, turanose, gentiobiose, cellobiose,
melezitose, xylitol, sorbitol, inositol, salicin,
adonitol , dulci tol, arabitol, erythritol, eryt hrose, raffinose, melibiose, fucose, t agatose,
lyxose, or sorbose.
Antibiotic susceptibilities: Susceptible to lysostaphin (MIC, 25 pglml), streptomycin (MIC,
3.1 pglml), erythromycin (MIC, 0.8 pg/ml), and
novobiocin (MIC, 0.1 pglml); slightly resistant
to penicillin G (MIC, 3.1 pglml); resistant to
tetracycline (MIC, 200 pg/ml); resistant to
lysozyme.
Cell wall peptidoglycan: L-LYs-G~Y,.,
, L
Ser,,. 2,
Cell wall teichoic acid composition: Glycerol
and galactosamine.
G+C content of the DNA: 34.0 mol%.
S . simulans can be distinguished from all
other staphylococci primarily on the basis of a
combination of its colony morphology, lack of
acetylmethylcarbinol production, carbohydrate
reaction pattern, and cell wall composition.
There are also some significant differences be-
TABLE
l l . Antibiotic susceptibilities of Staphylococcus species
Total
Species
I
no. of
strains
8
studied" o
Penicillin G
Novobiocin
Erythromycin
Tetracycline
Level of susceptibility*
1 Level of susceptibilit!
Level of susceptibility
Level of susceptibility
"?
y
8 2
zg &z G&+s3"A z F 4.;)
2-4- S T
40
0
"9,
zz
1 -
51
13
101
42
83
116
152
48
45'
46
2
20
8
26
2
49
4
8
23
47 25
91 7
95 5
<1 25
27 96
23 100
21
77
62 26
62 17
82 7
95
<1
-
72
-
96
1
98
3
0s JT+cq 3 -
d ~ o ~ d d d ~ o 5 % ? 8 ~
S . aureus . . . . . . . . . .
S . simulans . . . . . . . .
s. xylosus . . . . . . . . .
S. cohnii . . . . . . . . . . .
S . saprophyticus . . .
S . haernolyticus . . . .
S . warneri . . . . . . . . .
S . hominis . . . . . . . . .
S . epidermidis . . . . .
S . capitis . . . . . . . . . .
-
4 100
23 46
8
12 80
2
21 84 14
11 93
4
5 90 <1
100
64
83
85
46
18
2
3
9
36
17
15
-
Strains were isolated from persons who were not exposed to antibiotics for at least 3 months prior to collecting. It would be expected that the above base-level frequencies might shift toward higher frequencies of resistant strains during or shortly after antibiotic exposure (8).
bExpressed as MIC (numbers) and disk zone inhibition (numbers in parentheses). MICs are expressed as
micrograms per milliliter. Disk zone inhibition represents the width (in millimeters) of the zone of antibiotic
inhibition, measured from the edge of a 2-U penicillin G, 5-pg novobiocin, 2-pg erythromycin, and 30-pg
tetracycline sensitivity disk (Difco), respectively.
'Data are given as percentages of strains.
a
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S . aureus . . . . . . . . . . .
S . simulans . . . . . . . , ,
s. xylosus . . . . . . . . . .
S . cohnii . . . . . . . . . . . .
S . saprophyticus . . . .
S . haemolyticus . . , . .
S . warneri . . . . . . . . . .
S. hominis . . . . . . . . . .
S . epidermidis . . . . . .
S . capitis . . . . . . . . . . .
-
z
E!
:
3
Q
co
(CO)
(LCO)
(LCO)
(CO)
(CO)
RT
(RST)
RT
RT
4naerobic
growth in
thioglycolated
-
-
t
+
+
+
+
+
+
(+)
4.3-4.6
4.7-5.0
4.9-5.6
5.3-6.0
5.0-5.4
4.9-5.5
4.7-5.0
4.4-4.9
4.3-4.6
4.2-4.7
I'erminal
p H in
yeast
extractglucose
medium
Acid (aerobically)from
(T)
V
T
V
PS
PS
PS
V
V
(PS)
Cell
Colony
mange
profile
ment
and light
transmis- b e domision*
nantY
L
L>D
DL,D
DL
DL
D
DL,(L)
DL,L
L> D
L,DL
~
Lactic
acid
isomer(s
+
(*)
f
+
+
+
+
+
+
+
I
0.9-1.4
0.6-1.4
0.6-1.3
0.7-1.5
0.8-1.2
5.0-6.0
4.7-6.0
4.9-5.7
4 . 9 4 .0
4.1-5.1
3.3-4.0
3.3-4.4
3.3-4.3
4.0-5.0
3.5-4.3
5
R
.3
Q
-I----
-
-
Peptidoglycan
(moVmol of
glutamic acid)
-I
+
-
-
+
+
+
+
-
V
Teichoic acid
(components)
d
Cell-wall composition
V
f
(+)
+.
+
V
+
+**
f, -
+
(-)
-
+
1
I
Glutamic acid
polymer
(glutamic acid
2 1.3 mol/mol
lysine)
Coagu- Hemol.
Acety!- Nitrate
ribonuD~xyPhoslase
ysis
methyl- reducphatase
(pref.
(pref. rarbinol
tion
clease
rabbit) bovine1
(+)
(-)
+
+
Growth
at 45 C
Resistance to
Growth
on 10% ;rowth
NaCl 3t 15 C
agar
a
Characters were selected based on their ease of distinction and wide differences (270%) in their type frequencies between two or more species. A single listed symbol
denotes a type frequency of 90 to 100%; parentheses around symbols denote a type frequency of 70 to 89%; two symbols are listed for a character when either type is in
frequency below 70'70, but together equal 80 to 100%.Symbols for characters (unless noted otherwise): +, positive; i,weak; - , negative: V, variable ( + , f,and - ) .
Symbols for colony profile and light transmission: RT, raised (flat) and translucent; RST. raised (flat) and slightly translucent; CO, convex and opaque: LCO. low convex
and opaque.
Symbols for cell arrangement: PS, pairs and single cells; T, tetrads; V, variable (pairs, singles, and tetrads common).
Symbols for anaerobic growth: +. dense uniform; *. gradient of growth from dense to light down tube; +C, large individual colonies; c. small individual colonies to absence
of visible growth: V, variable (dense uniform, gradient growth, and individual colonies).
'Without exposure.
'ND, Not determined. (All of the strains were not tested. Some of the strains of S. xylosus and S . saprophyticus tested contained glucose. and most of the strains of
S . saprophyticus tested contained glucosamine.)
-
S . aureus . . . . . . . . . . . +
S . simulans . . . . , , , . . +
s. xylosus . . , . . . . . . . +
S . cohnii . . . . . . . . . . . . +
S . saprophyticus . . . . +
S . haemolyticus . . . . . +,S . warneri . . . . . . . . . . +
S . hominis . . . . . . . . . . +
S . epidermidis . . . :. . +
S . capitis . . . . . . . . . . . +
Species
6-8
5-7
5-9
5-7
5-8
4-8
3-5
3-4
2-4
1-3
Species
-
Colony
diameter
(mm)
TABLE
12. Scheme for the classification of human cutaneous staphylococcia
STAPHYLOCOCCI FROM HUMAN SKIN. 11.
VOL.25, 1975
77
Species colony morphology and pigment pattween S. simulans and certain other species in
the parameters of anaerobic growth in thio- terns are illustrated in Fig. 1. These characters
glycolate, lactic acid configuration, growth a t were very useful in making the preliminary
different NaCl concentrations and temperature species identification of strains on the original
extremes, coagulase activity, hemolysis, acetyl- isolation plates. Species having very distinctive
methylcarbinol production, nitrate reduction, colony characteristics include S. hominis, S.
and phosphatase, DNase, and bacteriolytic ac- warneri, S . epidermidis, and S . capitis. It was
tivities. Several of the properties of S . sirnulans somewhat difficult to differentiate a certain few
overlap with those of five unusual coagulase- strains of S. xylosus, S. saprophyticus, S .
positive strains isolated in this study that cohnii, and S . haemolyticus from one another
appear to be similar to strains previously de- using these characters alone.
It should be pointed out that the species
scribed by Reeder and Ekstedt (14)and Oeding
(12). There is also a n overlap of several proper- character variation observed in this study,
ties with those of human strains of s.aureus. It through very similar for the strains isolated from
would appear then that S . sirnulans is closely cities in North Carolina and New Jersey, may
be somewhat different with strains obtained
related to the coagulase-positive staphylococci.
Character variation in Staphylococcus from certain other geographical areas. For exspecies. Summary data of the morphological, ample, in this study we did not recover any of
physiological, biochemical, and antibiotic char- the violet-pigmented strains of S. epiderrnidis
acters found in the various Staphylococcus found by Marples (7) on people living in Philaspecies isolated from human skin are shown as delphia, Pa., and recently found by one of us
follows: colony morphology and pigment and (W.E.K.) and W. C. Noble (unpublished data)
cell arrangements, Table 5; anaerobic growth on people living in London, England. Strains
in thioglycolate, growth on 10% and 15% NaCl originating from animals other than man may
agar, and growth at 15 and 45 C, Table 6; also show somewhat different character variacoagulas e , he mol y sis , acety lm e t hy lcarbinol tion from the human strains.
Occurrence of Staphylococcus species on
production, nitrate reduction, phosphatase,
DNase, and bacteriolytic activities, Table 7; human skin. A summary of the occurrence and
carbohydrate reactions, Tables 8 and 9; lyso- distribution of Staphylococcus species on hustaphin susceptibility, Table 10; and antibiotic mans is shown in Table 13. As can be seen from
susceptibilities, Table 11. A summary scheme the data, some species show preferences for
showing the key characters used for the classifi- certain regions of the cutaneous habitat. S.
cation of cutaneous Staphylococcus species is epiderrnidis was a predominant species in all
presented in Table 12. This information is regions tested, including the head, nares, axilbased on species described in the companion lae, arms, and legs. S. hominis was a predominant species on the head, axillae, arms, and
paper (15) and this one.
TABLE
13. Occurrence of Staphylococcus species on human skin
% of 40
Species
S . aureus . . . . . . . . . .
S . simulans . . . . . . . .
s. xylosus . . . . . . . . .
S . cohnii . . . . . . . . . . .
S . saprophyticus . . .
S . haemolyticus . . . .
S . warneri . . . . . . . . .
S . epidermidis . . . . .
S . hominis . . . . . . . . .
S . capitis . . . . . . . . . .
individuals with
species
52
12
42
35
70
78
52
100
100
65
Mean 7%of skin sites occupied by
specieshndividual
Head Nares Axillae
Arms
Legs
4b
2
2
4
4
12
3
15
17
31
21
15
87
43
2
5
15
12
5
12
78
4
1
5
6
17
8
0
92
11
7
11
18
28
28
9
82
69
27
9
2
8
19
32
26
9
62
77
17
Mean % of species of total staphylococci (CFU)
recoveredsite per individual"
Head'
Nares
1.6 x 1 0 4 ~ 7.8 x lo6
5
1
4
3
3
4
1
46
22
11
30
0.1
0.06
0.3
0.1
0.3
0.3
63
5
0.8
Axillae
1.0 x 10'
0.02
0.05
0.2
0.2
0.3
0.9
0
64
34
0.3
* The number of colonies isolated from each site to determine the percentage of species present represents an
estimated 22 f 1%of the total staphylococcus colony-forming units (CFU) recoverable.
Number of sites per individual.
Average number of staphylococci per site per individual.
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78
KLOOS AND SCHLEIFER
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INT. J. SYST.BACTERIOL.
VOL.25, 1975
STAPHYLOCOCCI FROM HUMAN SKIN. 11.
legs, and to a lesser extent in the nares. S.
cupitis was a predominant species on the head
and to a lesser extent on the arms. S. uureus, as
expected from a considerable number of earlier
reports, was often a predominant species in the
nares.
ACKNOWLEDGMENTS
We are greatly indebted to Margaret Musselwhite for her
excellent technical advice and assistance throughout this
study. We are also grateful to E. Hagner, I. Pomper, and B.
Popp for their capable assistance. We are thankful for the
help provided by Merrill Emmett for maintaining and shipping cultures, Susan Kloos and Wesley L.Kloos for assisting
in collecting skin samples from persons living in New Jersey,
and Renee Johnson for assembling and typing tabular data
and assisting in preparing the manuscript. We also thank the
many people who provided us with samples of their skin flora.
This research was supported by Public Health Service
research grant A1 08255 from the Institute of Allergy and
Infectious Diseases and the Deutsche Forschungsgemeinschaft. Funds were provided by the North Carolina Agricultural Experiment Station for the color plate, which was
photographed by Ralph Mills and printed by Prolab, Raleigh,
N.C.
REPRINT REQUESTS
Address reprint requests to: Dr. Wesley E. Kloos, Department of Genetics, North Carolina State University, Raleigh,
N.C. 27607.
LITERATURE CITED
1. Auletta, A. E.,and E. R. Kennedy. 1966.Deoxyribonucleic acid base composition of some members of the
Micrococcaceae. J. Bacteriol. 92:28-34.
2. Baird-Parker, A. C. 1963.A classification of micrococci
and staphylococci based on physiological and biochemical tests. J. Gen. Microbiol. 30:409-427.
3. Baird-Parker, A. C. 1965.The classification of staphylococci and micrococci from world-wide sources. J. Gen.
Microbiol. 38:363-387.
4. Davis, G. H. G., and B. Hoyling. 1974.Observations on
79
anaerobic glucose utilization tests in StaphylococcusMicrococcus identification. Int. J. Syst. Bacteriol.
24:l-5.
5. Gordon, M. H. 1905. Report on bacterial test whereby
particles shed from the skin may be detected in the air.
Annu. Rep. Med. Officer Local Gov. Board (London)
5:387-409.
6. Kloos, W. E., T. G. Tornabene, and K. H. Schleifer. 1974.
Isolation and characterization of micrococci from
human skin, including two new species: Micrococcus
lylae and Micrococcus kristinae. Int. J. Syst. Bacteriol.
24~79-101.
7. Marples, R. R. 1969.Violagabriellae variant of Staphylococcus epidermidis on normal human skin. J. Bacteriol. 100:47-50.
8. Marples, R. R., and A. M. Kligman. 1971. Ecological
effects of oral antibiotics on the microflora of human
skin. Arch. Dermatol. 103:148-153.
9. Marples, R. R., A. M. Kligman, L. R. Lantis. and D. T.
Downing. 1970.The role of the aerobic microflora in the
genesis of fatty acids in human surface lipids. J. Invest.
Dermatol. 55:173-178.
10. Mitchell, R. G., V. G. Alder, and K. Rosendal. 1974.The
classification of coagulase-negative Micrococcaceae
from human and animal sources. J. Med. Microbiol.
7:131-135.
11. Noble, W. C. 1969.Skin carriage of the Micrococcaceae.
J. Clin. Pathol. 22:249-253.
12. Oeding, P. 1973. Wall teichoic acids in animal Staphylococcus aureus strains determined by precipitation. Acta
Pathol. Microbiol. Scand. Sect. B 81:327-336.
13. Perry, J. J. 1969.Isolation of Staphylococcus epidermidis
from Tobacco. Appl. Microbiol. 17:647.
14. Reeder, W.J., and R. D. Ekstedt. 1973.Unique teichoic
acid isolated from the cell walls of a strain of Staphylococcus aureus. Infect. Immunity 7586-588.
15. Schleifer, K. H., and W. E. Kloos. 1975. Isolation and
characterization of staphylococci from human skin. I.
Amended descriptions of Staphylococcus epidermidis
and Staphylococcus saprophyticus and descriptions of
three new species: Staphylococcus cohnii, Staphylococcus haemolyticus, and Staphylococcus xylosus. Int. J.
Syst. Bacteriol. 25:50-61.
16. Schleifer, K. H.,and M. Kocur. 1973. Classification of
staphylococci based on chemical and biochemical
properties. Arch. Mikrobiol. 93:65-85.
FIG. 1. Colonies of Staphylococcus species isolated from human skin. The “seed” inoculum was incubated at
34 C for 5 days (unless otherwise noted). (All rows read from left to right.) ( A ) S . aureus: top, GH 38; middle
row, KL 207, JL 68, KES 1; bottom row,GH 39, M A W306, HK 91 (Staphylococcus sp. related tolor a variety of
S . aureus). ( 3 )S . simulans: ATCC 27848 (MK Z#), ATCC 27849 ( AW 215), ATCC 27850 ( K L 240). ( C ) S .
xylosus: top row, A W 124, DM 37, A W244, GH 30; middle row, DSM 20266 ( K L 162),KH 168, TM 76, RM349;
bottom row,DBM 194, GH 9 (Staphylococcus sp. related tolor variety of S . xylosus). ( D )S . cohnii: DM 224,
DSM 20260 (GH 137),MK 247. ( E )S . saprophyticus: top row, TW111, K L 20, DM 100; bottom row, SE 1 1 , G H
l%, SM 118. (F)S . haemolyticus: top row, DM 70, TW268, GS 1 , MK 67; bottom row, KH 99, DSM2026.3 (SM
131), RM 419. ( G ) S . warneri: top row,MAW314, ATCC 27838 (MK 155),ATCC 27836 ( AW25);bottom row,
ATCC 27837 (RM 130),ATCC 27839 (DW 138), DM 229. ( H I ) S . hominis (incubated 5 days): top row,ATCC
27844 (DM 122), GH 180, LK 89, JM 16; bottom row, ATCC 27845 ( K L 2431, BS 14, ATCC 27847 (KES 131,
ATCC 27846 (MK 129). (H2)S . hominis (incubated 7days): colony identification same as above as in ( H l ) .( I )
S . epidermidis: top row, KL 275, MK 214, A W269; bottom row, RK 13, JRM 1 , KH 141, (4S . capitis: ATCC
27840 (LK 499), ATCC 27842 (MCS 1 1 ) , ATCC 27841 (DM 267).
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