IDENTITY OF

STAPHYLOCOCCUS
EPIDERMIDIS
Dorothy Jones, R. H. Deibel and C. F. Niven
Jr.
J. Bacteriol. 1963, 85(1):62.

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IDENTITY OF STAPHYLOCOCCUS EPIDERMIDIS'

DOROTHY JONES, R. H. DEIBEL, AND C. F. NIVEN, JR.
Division of Bacteriology, American Meat Institute Foundation, and The Department of Microbiology,
The University of Chicago, Chicago, Illinois
Received for publication 25 July 1962

In the classification of the family MicroEvans, Bradford, and Niven (1955)

proposed the separation of the genus Staphylococcus from Micrococcus on the basis of their
growth relationship to oxygen. These authors
suggested that the facultative species capable of
growing anaerobically in a standardized medium
containing glucose be placed in the genus Staphylococcus, and those species incapable of anaerobic
growth in the medium be assigned to the genus
Mlicrococcus. This distinction between Micrococcus and Staphylococcus has been incorporated
into Bergey's Manual (Breed, Murray, and Smith,
1957), where the genus Staphylococcus includes
two species, S. aureus and S. epidermidis.
Although S. aureus is a well-defined, homogeneous species, S. epidermidis includes a heterogeneous collection of mass-forming cocci sharing
the following characters: (i) ability to grow
anaerobically in a standardized medium containing glucose; (ii) inability to produce coagulase; and (iii) inability to ferment mannitol
anaerobically.
coccaceae,

Journal

paper

234, American Meat Institute

Foundation.
62

The present paper attempts to provide a more

definitive characterization of the species S.
epidermidis. In addition, the taxonomic position
of Al. hyicus [a pathogenic, coagulase-negative,
catalase-positive coccus isolated by Sompalinsky
(1950, 1953), and shown by him to be the etiological agent in an outbreak of contagious impetigo
of swine] was investigated and compared with S.
epidermidis.
MATERIALS AND METHODS
Source of strains. The majority of the strains
employed in this study were isolated from food
products, and from a variety of clinical sources.
One strain of Al. hyicus was obtained from D.
Sompalinsky, Institut de Serologie Veterinaire
de l'Etat, Copenhagen.
Physiological methods. Most of the physiological tests have been described previously (Evans
and Niven, 1950). Carbon dioxide production
from glucose was determined qualitatively by the
method of Williams and Campbell (1951).
In experiments concerned with the anaerobic
utilization of the various substances as energy
sources, the basal medium consisted of Tryptone
(Difco), 10 g; yeast extract (Difco), 5 g; K2HPO4,
5 g; NaCl, 5 g; energy source, 10 g; distilled water,
1 liter; pH 7.0 to 7.2. For anaerobiosis, broth
cultures were placed in a 6-liter desiccator and
flushed twice with hydrogen. The final gas phase
consisted of a mixture of 90% hydrogen and 10%
carbon dioxide. Growth was estimated by determining the optical density of cultures in a Spectronic 20 spectrophotometer at a wavelength
setting of 600 m,.
Anaerobic requirement for uracil was determined in the casein hydrolysate medium of
Gretler et al. (1955). Uracil was added to give a
final concentration of 1 mg/100 ml of medium.
Biotin requirement was determined in Biotin
Assay Medium (Difco). The egg yolk opacity
test was detected on Colbeck EY agar plates
(Difco), and deoxyribonucleic acid hydrolysis determined by the method of Di Salvo (1958).

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ABSTRACT
JONES, DOROTHY (American Meat Institute
Foundation, Chicago, I11.), R. H. DEIBEL, AND
C. F. NIVEN, JR. Identity of Staphylococcus
epidermidis. J. Bacteriol. 85:62-67. 1963.-The
species Staphylococcus epidermidis is defined
more precisely, and compared with other staphylococci. The physiological characteristics considered to be of primary import are: (i) ability to
grow anaerobically in a defined complex medium
with glucose or pyruvate as the energy source;
(ii) inability to produce coagulase; (iii) ability
to ferment serine as an energy source; (iv) the
requirement for biotin and uracil (under anaerobic conditions) in a semisynthetic medium; and
(v) inability to reduce nitrate beyond nitrite.

VOL. 85, 1963

IDENTITY OF STAPHYLOCOCCUS EPIDERMIDIS

63

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TABLE 1. Physiological characteristics of the

RESULTS
strains tested
General physiological characterization. Strains
(53) of coagulase-negative, catalase-positive,
Organism and no. of strains tested
gram-positive cocci were screened for their ability
Physiological
MicroDenitriStaphyloto utilize glucose as a source of energy under
characteristic
coccus
coccus
fying
anaerobic conditions. Of the strains, 43 grew
epidermidis hyicus staphylo(15)
(1)
cocci (2)
under these conditions, and were classified
tentatively as S. epidermidis (Breed et al., 1957); Anaerobic glucose
17 of these strains, together with the 1 strain of
utilization ......
+
Ml. hyicus, were selected for a more complete Coagulase ..........
+
physiological characterization. Six or more Nitrate reduction... II+ 4strains of S. aureus were included for comparative Pigmentation
+
Yellow............ 1+
purposes.
None
.............
14+
All of the strains utilized glucose as a source of
energy under anaerobic conditions (Table 1). Growth
10 C .............. 1+, 14- +
Consequently, M. hyicus would be termed more
45 C.............. 9+, 61+, 1appropriately Staphylococcus hyicus. However, Hydrolysis
for purposes of presentation, the more common
Starch ............
generic name associated with this species will be
Sodium hippurate.
used.
Esculin ........... 3+, 12Three of the strains (Al. hyicus and StaphyArginine .......... 14+, 1Gelatin ........... 13+, 2lococcus strains H60 and H92) reduced nitrate
beyond nitrite. This characteristic merits separa- Peptonization, litmus milk....... 11+, 4tion of these strains from the typical S. epidermidis. In addition, all of these strains failed to Acetoin from glu............ 14+, 1produce acetoin, and fermented glucose to a CO2 cose
+
from glucose.... +
1+, 1lower final pH value than the S. epidermidis Fermentation*
(acid)
strains. In the following discussion, StaphyMannitol (aero+
lococcus strains H60 and H92 will, therefore, be
bic) .............
1+, 1+
referred to as "denitrifying" staphylococci.
Mannitol (anaerobic) .............
The majority of the S. epidermidis strains, the
Mannose ..........
1+, 1two denitrifying strains, and M. hyicus formed a
Galactose ......... 11+, 4rennetlike curd in litmus milk (Difco), and upon
Maltose ........... +
1,4-4.
further incubation this curd was digested slowly.
Lactose ........... +
This reaction in litmus milk has not been reported
Trehalose ......... 1+, 13previously for S. epidermidis (Breed et al., 1957).
Glycerol .......... 8+, 7No distinct fermentation pattern was detected
Melezitose ........ 2+, 13in the S. epidermidis group. Although the anaero- Final pH, glucose... 5.2-5.7 5.5
bic fermentation of mannitol is usually associated
* All
strains produced acid from glucose, frucwith S. aureus, one of the denitrifying staphyand sucrose. None of the strains evidenced
tose,
lococci fermented this alcohol. Maltose was not
fermented by the denitrifying staphylococci, nor acid production from xylose, arabinose, raffinose,
sorbitol, dextrin, salicin, melibiose, or
by Mll. hyicus, although all of the S. epidermidis inulin,
cellobiose.
strains fermented this substrate. Sompalinsky
(1950, 1953) stressed inability to ferment maltose
strains included in this study produced opacity
in his description of M. hyicus.
24 hr. In contrast, the S. epidermidis
within
Hydrolysis of deoxyribonucleate. Only S. aureus
exhibited
a weak and delayed (48 to 72 hr)
strains
and M. hyicus evidenced appreciable deoxyribonuclease activity. Although M. hyicus demon- positive reaction, and M. hyicus was devoid of
strated weaker enzyme activity than S. aureus, activity.
Anaerobic utilization of pyruvate. In recent
its reaction was decidedly positive.
it has been observed that the dismutation
aureus
of
S.
reaction.
All
the
years,
Egg yolk opacity

JONES, DEIBEL, AND NIVEN

64

TABLE 3. Typical growth responses (optical density

X 100) of staphylococci with pyruvate and glucose
as anaerobic energy sources*
Organism and strain

Staphylococcus epidermidis
Fussell ...................
NU13B ...................
B5B .....................
3RB2 .....................
Micrococcus hyicus .......
Denitrifying staphylococci
H60 .....................
H92 .....................
S. aureus
43 ......................
OB14 .....................
170 .....................
CB8 .....................

Substrate
Pyruvate
Glucose

119
116
122
135
90
....64
..

70
80
110
119

97
107

122
113

122
140
130
122

37
110
90
77

* The basal medium is described under Materials and Methods. Results determined after 24
hr of incubation at 37 C.

served when the S. epidermidis strains were

cultured with pyruvate as the energy source.
Quite often this material interfered with the
optical-density determination of the growth
response. No slime was detected when the strains
were cultured with other energy sources. Deibel
(1962) reported slime production by S. faecalis
in a pyruvate medium. The chemical nature of
this viscous material will be the subject of a
future publication.
The utilization of L-serine as an energy source
by S. epidermidis paralleled closely the utilization of pyruvate (Table 4). However, the majority of the S. aureus strains were unable to
derive energy from this amino acid. Although the
TABLE 2. Anaerobic utilization of glucose and
mechanistic aspects of serine degradation were
pyruvate by various Staphylococcus strains*
not investigated, it is assumed that a deamination (serine dehydrase) takes place with the
No. of
strains Glucose Pyvtr
Organism
formation of pyruvate. Characteristically, cultested
tures evidencing growth with serine produced
large quantities of ammonia.
+
+
Staphylococcus epidermidis. 41
S.
10
+
+
Although the vast majority of the strains emDenitrifying staphyloployed in this study hydrolyzed arginine, only
cocci
2
+
+
one of the two denitrifying strains, M. hyicus,
1
Micrococcus hyicus
+
+
and
one S. epidermidis strain were able to utilize
Micrococcus sp
10
this amino acid as a source of energy (Table 4).
*
Malate, citrate, gluconate, ascorbate, and
The basal medium is described under Materials and Methods. Results determined after 24 glycerol were also tested for their ability to serve
hr of incubation at 37 C.
as energy sources for the staphylococci under
vate

aureus

.........

.............

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of pyruvate is an energy-yielding process in which

lipoic acid plays a key role (Gunsalus, 1954).
More recently, Deibel, Evans, and Niven (1958)
and Deibel and Niven (1960) reported that the
anaerobic utilization of pyruvate, citrate, serine,
malate, and arginine (Deibel, 1960) as energy
sources can be used to distinguish Streptococcus
faecalis from Streptococcus faecium. S. faecalis
utilized these substrates, and, furthermore,
growth with pyruvate, citrate, and serine as the
energy source required an exogenous source of
lipoic acid.
The entire Staphylococcus-Micrococcus collection was tested for the ability to utilize pyruvate
as a source of energy under anaerobic conditions.
For comparative purposes, the growth response
with glucose was determined. All of the S. epidermidis, S. aureus, denitrifying staphylococci, and
M. hyicus strains utilized pyruvate and glucose,
whereas none of the aerobic Micrococcus strains
was capable of growing under these conditions
(Table 2). Worthy of mention are the relative
growth responses of the various groups to pyruvate and glucose (Table 3). S. epidermidis, the
denitrifying staphylococci, and M. hyicus exhibited equal or usually higher optical densities
with pyruvate as the energy source. In contrast,
the S. aureus strains demonstrated a superior
growth response with glucose as the energy
source, although they did evidence a significant
growth response with pyruvate. Unlike Streptococcus faecalis (Deibel et al., 1958), no exogenous
lipoic acid requirement could be demonstrated
with the S. epidermidis strains when tested in a
casein hydrolysate, semisynthetic medium (Deibel, 1962).
A high incidence of slime production was ob-

J. BACTERIOL.

IDENTITY OF STAPHYLOCOCCUS EPIDERMIDIS

VOL. 85, 1963

TABLE 4. Utilization of serine and arginine as

energy sources by staphylococci*
Substrate

Sta phylococcus
aureus

(9)

S. epider(15)
midis

Micrococcus
hyicus
(1)

Denitrifying
staphylococci (2)

65

staphylococci at one extreme and the thermoduric

saprophytes at the other. Shaw, Stitt, and Cowan
(1951) placed the entire group in the one genus,
Staphylococcus. These authors recognized five
species: S. aureus, S. saprophyticus, S. lactis, S.

roseus, and S. afermentans. Hill (1959), using the

Adansonian method of classification (Sneath,
+
+
+
+
1957), agreed with the conclusions of Shaw et al.
Serine
+
3+ 6- 14+, 1- (1951) regarding S. aureus and S. saprophyticus,
1+, 14- + 1+, 1- but was of the opinion that S. lactis and S.
Arginine
* The basal medium is described under Materials afermentans were too heterogeneous a collection
and Methods. Results determined after 24 hr of to warrant species status. In addition, using the
method of Evans et al. (1955), Hill preferred to
incubation at 37 C.
reclassify Staphylococcus roseus as Micrococcus
anaerobic conditions. With the exception of one roseus.
Pohja (1960), in an exhaustive investigation of
strain of S. aureus which utilized citrate, malate,
and ascorbate, none of the other organisms the little-studied meat Micrococcus-Staphylococcus
group, placed the coagulase-positive strains (S.
studied utilized these compounds.
Nutritional studies. Richardson (1936) demon- aureus) and the acetoin-producing strains (S.
strated an absolute requirement for uracil by saprophyticus; Shaw et al., 1951) in the genus
some strains of S. aureus when grown under Staphylococcus, and strains which he could not
anaerobic conditions. This requirement has never classify in this way were assigned to the genus
been exploited fully in any taxonomic study Micrococcus. Consequently, this genus as proundertaken with the Staphylococcus-Micrococcus posed by Pohja, unlike the genus Micrococcus as
group; 93% of the S. epidermidis strains required defined by Evans et al. (1955), includes aerobic
uracil for anaerobic growth (Table 5). The two and facultative anaerobic organisms.
In the present study, a primary separation of
denitrifying strains manifested no uracil requiretypes was effected by employing a
physiological
or
when
uracil,
ment; M. hyicus did not grow
the method of Evans et al. (1955).
of
modification
uracil in combination with guanine and adenine,
The
anaerobic
growth response to glucose was
was added to the basal medium. The additional
a sharp differentiaturbidimetrically;
estimated
requirements of this strain were not investigated.
In addition,
in
manner.
this
be
effected
tion
could
et
al.
of
Gretler
the
results
(1955),
Confirming
as a source
of
utilization
the
anaerobic
pyruvate
all of the coagulase-negative staphylococci rewith
obtained
results
the
of
energy
paralleled
quired the vitamin biotin for growth (Table 5).
glucose, and thus emphasized the validity of this
DISCUSSION
primary differentiation of physiological types in
The classification of the aerobic and facultative TABLE 5. Biotin and uracil (anaerobic) requirements
anaerobic strains of the family Micrococcaceae is
of staphylococci*
far from satisfactory. The literature reflects this
DenitriMicroStaphylounsatisfactory condition in the plethora of generic
stpyincoccus
S. epidercoccus
tan ce
(s
t subs
nitaphylmidis1der
sustaceaureus
(15) hyicus
and specific epithets that have been and still TesTes
(1)
COCCi2
the
be
termed
what
can
are employed to classify
Micrococcus-Staphylococcus group. Although some
+
14+, 1- +
progress has been made in the direction of a more Uracil
(anaerorational and universally acceptable classification,
bic)
the central question of whether this group of
+
+
+
_
microorganisms should be included in one genus Biotin
or separated into two genera still is not resolved
*
Readings made after 24 hr of incubation at
completely. Moreover, the composition of these 37 C. The semisynthetic media are described
genera remains in a fluid state.
under Materials and Methods. Symbols: +, reAbd-el-Malek and Gibson (1948) regarded the quirement; -, no requirement; i, additional,
entire group as a linear series, the pathogenic undetermined requirement.

Pyruvate
(anaerobic)

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oc

66

JONES, DEIBEL, AND NIVEN

trifying staphylococci warrant separation of

these microorganisms from S. epidermidis, their
exact taxonomic position must await further
study. The preliminary results obtained with
these strains indicate that they may differ sufficiently from other staphylococci to merit species
rank.
S. epidermidis as defined by Breed et al. (1957)
appears to be synonymous with S. saprophyticus
as described by Hill (1959). However, two strains
of S. saprophyticus (NCTC 7292 and 7612)
obtained from S. Cowan, Central Public Health
Laboratory, Colindale, London, failed to grow
anaerobically when provided with glucose or
pyruvate as the energy source. These results
indicate the necessity for further studies with this
species prior to determining its exact taxonomic
position.
ACKNOWLEDGMENTS

The authors wish to extend appreciation to

P. T. Fagan, St. James Clinical Laboratories,
Chicago Heights, Ill., for the use of his laboratory
in the isolation of the clinical strains employed
in this study.
This work was supported in part by grant
E-1951 from the National Institutes of Health,
U.S. Public Health Service.
LITERATURE CITED
ABD-EL-MALEK, Y., AND T. GIBSON. 1948. Studies
in the bacteriology of milk. II. The staphylococci and micrococci of milk. J. Dairy Res.

15:249-260.
BREED, R. S., E. G. D. MURRAY, AND N. R.
SMITH. 1957. Bergey's manual of determinative bacteriology, 7th ed. The Williams &
Wilkins Co., Baltimore.
DEIBEL, R. H. 1960. Arginine as an energy source
for the growth of Streptococcus faecalis.
Bacteriol. Proc., p. 163-164.
DEIBEL, R. H. 1962. Utilization of pyruvate as an
energy source for Streptococcus faecalis.
Thesis, The University of Chicago, Chicago.
DEIBEL, R. H., J. B. EVANS, AND C. F. NIVEN,
JR. 1958. Lipoic acid requirement for anaerobic
utilization of pyruvate as an energy source
by Streptococcus faecalis. Bacteriol. Proc.,
p. 114.
DEIBEL, R. H., AND C. F. NIVEN, JR. 1960. Lipoic
acid and the fermentation of serine and malate

by Streptococcus faecalis. Bacteriol. Proc.,

p. 164.

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the Micrococcus-Staphylococcus group. Moreover,

the observation that some of the facultative
anaerobic strains reduced nitrate beyond nitrite
further aided in delineating the remaining group.
The inability of S. aureus to ferment pyruvate
with facility detracts from the possible use of this
character to define this species, although it may
well be incorporated in the description of S.
epidermidis. The fermentation of serine, which is
presumably deaminated to pyruvate, may also
be employed to define S. epidermidis.
The anaerobic requirement for uracil may also
prove to be of taxonomic value. All the strains of
S. aureus employed in this study, and all but
one of the strains of S. epidermidis tested, required uracil. However, the denitrifying staphylococci and M. hyicus either exhibited no such
requirement or else required an additional growth
supplement, the nature of which was not determined.
Richardson (1936) investigated six strains of
S. aureus, and four grew anaerobically when only
uracil was added. The other strains grew only if
hydrolyzed nucleic acid was added.
Acetoin production has long been considered
an important characteristic within this group of
microorganisms (Abd-el-Malek and Gibson, 1948;
Shaw et al., 1951; Hill, 1959). In this study, one
strain, classified by us as S. epidermidis on the
basis of other criteria, produced acid from glucose
but failed to produce acetoin. The two denitrifying strains reacted similarily, but all these strains
produced acetoin from pyruvate. M. hyicus,
however, produced acetoin neither from glucose
(confirming the findings of Sompalinsky) nor
from pyruvate.
On the basis of its ability to utilize glucose and
pyruvate anaerobically, a case can be made for
the reclassification of M. hyicus as a Staphylococcus. This organism possesses a unique set of
characteristics in that it is a coagulase-negative,
pathogenic Staphylococcus. Its pathogenicity was
established previously in pigs by Sompalinsky
(1950, 1953) and confirmed in this laboratory
in mice. In the mouse, necrotic skin lesions develop after 3 to 5 days when the organism is
injected subcutaneously. Although the property
of pathogenicity alone may not contribute to
species status, Staphylococcus hyicus differs sufficiently from S. epidermidis to remain outside
this species.
Although the results obtained with the deni-

J. BACTE:RIOL.

VOL.

85,

1963

IDENTITY OF STAPHYLOCOCCUS EPIDERMIDIS

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Di

67