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lnternational Journal
of
Systematic Bacteriology
(1
998), 48,859-877
Printed in Great Britain
1
North Carolina State
University, Raleigh,
NC
27695-7614, USA
2
Qualicon Inc., a DuPont
subsidiary, Wilmington,
DE
19880-0402, USA
3
Technische Universitat
Milnchen, 80290 Munich,
Germany
80638
Munich, Germany
4
Universittit
MOnchen,
Delimiting the genus
Staphylococcus
through
description
of
Macrococcus caseolyticus
gen.
nov.,
comb.
nov.
and
Macrococcus equipercicus
Spm
nov.,
Macrococcus bovicus
sp. nov. and
Macrococcus carouselicus
sp.
nov.
Wesley
E.
KIoos,' Deborah
N.
Ballard,l Carol G. George,'
John
A.
Webstert2 Romeo
J.
Hubner,2 Wolfgang Ludwigf3
Karl
H.
S~hleifer,~ Franz FiedleP and Karin Schubert4
Author for correspondence: John A. Webster. Tel:
+
1
302
695 1613.
Fax:
+
1
302
695 8557.
e-mail
:
john.a.webster @usa.dupont .corn
Four
species
of
the newly proposed genus Macrococcus, namely Macrococcus
caseoiyticus gen. nov.,
comb.
nov. (formerly Staphy/ococcus
caseo/yticus
Schleifer, Kilpper-Balz, Fischer, Faller and
Endl
1982,
1
SVp),
Macrococcus
equipercicus sp. nov., Macrococcus bovicus sp. nov. and
Macrococcus
carouselicus sp. nov., are described
on
the basis
of
a phylogenetic analysis
comparing
165
rRNA sequences, DNA-DNA
liquid
hybridization, DNA base
composition, normalized ribotype patterns, macrorestriction pattern analysis
and estimation
of
genome size using PFGE, cell wall composition, phenotypic
characteristics and plasmid profiles. Compared
with
their closest relatives,
members
of
the genus Staphylococcus, these organisms demonstrated
significantly lower
165
rRNA sequence similarities
(934-9503
%),
higher DNA
G+C content
(38-45
mol
YO),
absence
of
cell wall teichoic acids (with the
possible exception
of
M.
caseolyticus), unique ribotype pattern types and
macrorestriction patterns, smaller genome size (approx.
1500-1800
kb)
and
generally larger Gram-stained cell size
(14-205
pm
in
diameter). Macrococci can
be distinguished
from
most species
of
staphylococci (except Staphylococcus
sciuri, Staphylococcus witulus and Staphylococcus lentus)
by
their oxidase
activity. The
four
Macrococcus species can be distinguished
from
one another
on
the basis
of
DNA-DNA hybridization, ribotype pattern types,
macrorestriction patterns and their phenotypic properties, including colony
morphology,
cell morphology, haemolysins, Staph Latex agglutination, acid
production
from
a variety
of
carbohydrates, acetoin
production,
nitrate
reduction, aesculin hydrolysis, and DNase and urease activities. The type
species is
M.
equipercicus. The type strains
of
M.
equipercicus,
M.
caseo/yticus,
M.
bovicus and
M.
carouselicus are ATCC
5183IT
(=
DD
93503,
ATCC
13548l
(=
TDD
45083
(Schleifer eta/.
1982),
ATCC
51825l(=
DD
45163
and ATCC
51828T
(=
DD
93483,
respectively.
Keywords:
Macrococcus
gen. nov.,
Macrococcus
caseolyticus
gen. nov.,
comb.
nov.,
Macrococcus equipercicus
sp.
nov.,
Macrococcus
bovicus
sp.
nov.,
Macrococcus carouselicus
sp.
nov.
Abbreviation:
SEM,
scanning electron microscopy.
The
EMBL
accession numbers
for
the
165
rRNA sequences
of
M.
caseolyticus ATCC
13548T,
M.
equipercicus ATCC
51831T,
M.
carouselicus ATCC
5lS2ST
and
M.
bovicus
ATCC
5182ST
are
Y15711-Yl5714,
respectively.
00683
0
1998
IUMS
859
W.
E.
Kloos
and others
INTRODUCTION
Comparative chemical, biochemical and nucleic acid
hybridization analyses of strains designated as
'
Micro-
coccus caseolyticus
'
ATCC 13548 and
Micrococcus
varians
ATCC 29750 indicated that they are related to,
but distinct from, other known members of the genus
Staphylococcus,
and for this reason they were placed in
a new staphylococcal species named
Staphylococcus
caseolyticus
(Schleifer
et al.,
1982). These strains were
isolated from the milk of cattle, but more recently
several strains have been isolated from the abscesses of
slaughtered lambs (De La Fuente
et al.,
1992) and one
from the milk of goats (De Buyser
et al.,
1992).
In a preliminary investigation (Ballard
et al.,
1995), we
sampled the skin of 15 cattle, 25 goats, 14 horses, 10
ponies,
4
whales,
25
dolphins and meat products for
the presence of
Staphylococcus caseolyticus.
This spe-
cies was isolated from only three samples of raw beef
and the skin of a pilot whale and
so
can still be thought
of
as a relatively uncommon species. Surprisingly, we
discovered a group of three new species related to
Staphylococcus caseolyticus
living on the skin of cattle,
horses and ponies. The group was found as large
populations on 11 of the 14 horses and all of the ponies
sampled in this study. This report is a description of
these organisms based on a variety of phenotypic
characters, cell wall composition, electrophoretic pat-
terns
of
EcoRI
restriction fragments labelled by
hybridization with an rRNA operon from
Escherichia
coli,
macrorestriction patterns of SmaI and
Not1
chromosome fragments, DNA-DNA liquid hybrid-
ization, DNA base composition and
16s
rRNA
sequences. Compared with staphylococci, these organ-
isms demonstrated significantly lower 16s rRNA
sequence similarities, higher DNA
G
+
C content,
absence of cell wall teichoic acids, larger cell size and
smaller genome size, indicating that the new species
and
Staphylococcus caseolyticus
should be placed into
a separate, but related genus, which has been given the
name
Macrococcus
gen. nov.
METHODS
Bacterial strains.
The strains selected for the descriptions of
Macrococcus caseolyticus
(Schleifer
et al.
1982) gen. nov.,
comb. nov.,
Macrococcus equipercicus
sp. nov.,
Macrococcus
bovicus
sp. nov. and
Macrococcus carouselicus
sp. nov.,
including their designations and sources, are listed in Table
1. Unless indicated otherwise, strains were isolated from the
skin of mammalian hosts. The following type strains of
staphylococci, bacilli and salinicocci were used for making
various comparisons with macrococci
:
Staphylococcus vitu-
lus
ATCC 51145T
(=
DD 756T),
Staphylococcus lentus
ATCC 29070T
(=
DD 4203T),
Staphylococcus
sciuri
subsp.
sciuri
ATCC 29062T
(=
DD 4277T),
Staphylococcus muscae
CCM 4175'
(=
DD 4795T),
Staphylococcus hyicus
ATCC
1
1249T
(=
DD 887T),
Staphylococcus chromogenes
ATCC
43764T
(=
DD 851T),
Staphylococcus gallinarum
ATCC
35539T
(=
DD 854T),
Staphylococcusequorum
ATCC 43958T
(=
DD 853T),
Staphylococcus kloosii
ATCC 43959T
(=
DD
857T),
Staphylococcus auricularis
ATCC 33753T
(=
DD
872T),
Staphylococcus aureus
ATCC
I
2600T
(
=
DD 10 1 56T),
Staphylococcus epidermidis
ATCC 14990T
(=
DD 8726'),
Staphylococcus hominis
ATCC 27844T
(
=
DD 2708T),
Staph-
ylococcus haernolyticus
ATCC 29970T
(=
DD 85ST>,
Staphylococcus capitis
ATCC 27840'
(
=
DD 873'),
Staphyl-
ococcus saccharolyticus
ATCC 14953*
(=
DD 860T),
Staph-
ylococcus saprophyticus
ATCC 1 5305T
(
=
DD 866T),
Staphylococcus warneri
ATCC 27836T (DD
=
870T),
Staphylococcus schleiferi
subsp.
schleiferi
ATCC 43808'
(
=
DD 86IT),
Staphylococcus intermedius
ATCC 29663T
(=
DD 856T),
Staphylococcus carnosus
DSM 20501T
(=
DD
1091'),
Bacillus subtilis
ATCC
6051T
(=
DD 379'),
Bacillus
subtilis
I68
and
Salinicoccus roseus
ATCC 49258T
(=
DD
1
2047T).
Phenotypic characterization.
The following characteristics
were determined as described previously (Kloos
&
Schleifer,
1975; Schleifer
&
Kloos, 1975; Kloos
et al.,
1976; Webster
et
al.,
1994; Kloos
&
Bannerman, 1995): Gram-stained cell
morphology and cell arrangement, colony morphology and
pigmentation, motility, anaerobic growth in thioglycollate
semi-solid medium, catalase activity, acetylmethylcarbinol
(acetoin) production, nitrate reduction, oxidase activity,
pyrrolidonylarylamidase activity, aesculin hydrolysis,
DNase activity, thermonuclease activity, ornithine de-
carboxylase activity, urease activity, staphylocoagulase ac-
tivity, lysostaphin susceptibility, haemolysis of sheep, bovine
and horse blood, and carbohydrate reactions. The presence
of
clumping factor and/or protein A was tested using the
Staph Latex Kit (Remel). Antibiotic susceptibilities were
determined by disk-diffusion testing, involving the incu-
bation of cultures on tryptic soy agar (TSA; Difco) and
Mueller-Hinton agar plates at
35
"C for 24 h in the presence
of various antibiotic disks, followed by the measurement
of
diameters
of
zones of inhibition (haloes) (Woods
&
Washin-
gton, 1995). Colony size, pigmentation, lustre, profile and
consistency were determined on freshly prepared
P
agar
(Kloos
et al.,
1991), TSA, and TSA plus
5%
sheep blood
plates following point inoculation, incubation for 72 h at
35 "C and storage at room temperature for an additional
2
d
(Kloos
&
Schleifer, 1975; Kloos
&
Bannerman, 1995).
Alkaline phosphatase, urease, P-galactosidase, P-glucosidase
and P-glucuronidase activities, and arginine utilization were
tested with the API STAPH-IDENT system (bioMerieux).
Additional biochemical profile data were obtained by using
the STAPH Trac and ID32 STAPH systems (bioMerieux
Vitek).
Normalized ri
botyping.
Discrete and reproducible ribotype
descriptions of strains resulted from a single method
described previously in detail (Webster
et al.,
1994; Bruce
et
al.,
1995; Hubner
et al.,
1995). Briefly, strains were lysed
with staphylolytic enzymes and SDS. The resulting solution
was extracted with phenol and chloroform, and the DNA
was precipitated with ethanol. The DNA was digested with
EcoRI. The resulting fragments were separated using aga-
rose-gel electrophoresis in a minigel apparatus. The size-
separated fragments were immobilized on a membrane by
electrophoretic transfer, denatured and probed. The
rrnB
ribosomal operon from
E.
coli
(Brosius
et al.,
1981), labelled
by sulfonation, was used as a probe. Immobilized fragments
selected by hybridization were detected with an anti-
sulfonated DNA monoclonal antibody conjugated to al-
kaline phosphatase and a chemiluminescent substrate.
Images of bands on the membrane were captured elec-
tronically and recorded. Computerized procedures were
860
International
Journal
of
Systematic Bacteriology
48
Macrococcus
gen. nov.
used to extract the strain data from each lane and digitally
process the bands to a normalized and reproducible charac-
terization. Each strain was then represented in a database by
a set of fragment bands. Sets from independently isolated
strains that were indistinguishable within the experimental
error were averaged and the mean value was stored as a
ribotype pattern type.
Genomic DNA-DNA hybridization.
Unlabelled and
[methyl-
3H]thymidine-labelled total DNAs were isolated and puri-
fied by using a modification of the procedures of Brenner
et
al.
(1969) for use with
Macrococcus
and staphylococcal
species that are somewhat difficult to lyse (Kloos
&
Wolf-
shohl, 1979; Kloos
et al.,
1997). DNA-DNA reassociation
reactions were performed in duplicate or triplicate under
stringent (70 "C) and optimal
(55
"C) conditions (Kloos
&
Wolfshohl, 1979; Kloos, 1980, 1998). At the termination of
reactions, double-stranded (hybridized) DNA was separated
from unreacted, single-stranded DNA on hydroxyapatite by
the batch procedure described by Brenner
et al.
(1 969). The
relative binding (or DNA relatedness) was determined by
normalizing the amount of DNA bound to hydroxyapatite
in heterologous reactions to that bound in the homologous
reaction, where labelled and unlabelled DNA were from the
same organism.
Phylogenetic analyses based on
165
rRNA sequences.
The
procedures for determining the 16s rRNA sequences of the
Macrococcus
species type strains have been described pre-
viously (Springer
et al.,
1993). The new 16s rRNA sequences
were added to an alignment of homologous primary struc-
tures available from public databases (Maidak
et al.,
1996;
Van de Peer
et al.,
1996). Phylogenetic analyses were
performed by applying distance matrix, maximum-parsi-
mony and maximum-likelihood methods on different data
sets. Distance matrix as well as maximum-parsimony analy-
ses were performed using data sets comprising all available
16s rRNA primary structures from Gram-positive bacteria
with a low DNA G
+
C content as well as reference sequences
from selected representatives from each of the remaining
major phylogenetic groups of bacteria. Maximum-likeli-
hood methods (Spring
et al.,
1996) were used to analyse
subsets of about
50
sequences comprising the homologous
primary structures from macrococci, staphylococci,
B.
subtilis
and
Salinicoccus roseus,
and also selected outgroup
references from other major phylogenetic groups. The data
sets varied with respect to the reference sequences as well as
the alignment positions. The variabilities of the individual
alignment positions were determined and used as criteria to
successively remove highly variable positions from the data
set. This was done to recognize and minimize treeing
artefacts resulting from alignment errors, database incon-
sistencies and
'
false
'
identities (multiple base changes) in
highly variable regions. The calculations and data analyses
were performed using the
ARB
program package (Ludwig
&
Strunk, 1997).
Nucleotide sequence accession numbers.
The EMBL ac-
cession numbers for the 16s rRNA sequences used in
addition to those of the four macrococcal species are:
Staphylococcus sciuri
ATCC 29062T, S83569
;
Staphylo-
coccus muscae
CCM 41 75T, S83566;
Staphylococcus auricu-
laris
ATCC 33753T, D83358;
Staphylococcus aureus
ATCC
12600T, X68417, L37599
;
Staphylococcus epidermidis
ATCC
14990T, D83363
;
Staphylococcus hominis
ATCC 27844T,
X66 10 1, L3760 1
;
Staphylococcus haemoly ticus
ATCC
29970T, D83367, L37600
;
Staphylococcus capitis
ATCC
27840T, L37599
;
Staphylococcus saccharolyticus
ATCC
1
4953T, L37602
;
Staphylococcus saprophyticus
ATCC
1 5305T, L37596
;
Staphylococcus warneri
ATCC 27836T,
L37603
;
Staphylococcus schleiferi
ATCC 43808T, S83568
;
Staphylococcus intermedius
ATCC 29663T, D83369
;
Staphylococcus carnosus
DSM 20501T, 226891
;
B.
subtilis
168, K00637, M10606, X00007
;
and
Salinicoccus roseus
ATCC 49258T, X94559.
PFGE
of
chromosome fragments.
DNA was prepared ac-
cording to a modification of the procedures of George
&
Kloos (1994). Cultures were grown in
5
ml Brain Heart
Infusion (BHI; Difco) broth for 18
h
at
35
"C. A
1
ml
aliquot of the overnight culture was then added to a 1.5 ml
microcentrifuge tube and centrifuged for
5
min in a Micro-
Centrifuge (model 235A: Fisher Scientific). The cell pellet
was resuspended in
1
ml TEN buffer (0-1 M Tris, 0.15 M
NaCl, 0.1 M EDTA
,
pH 7.5) and centrifuged again for
5
min. The resulting cell pellet was resuspended in 300 pl EC
lysis buffer (6mM Tris/HCl,
1
M NaC1, 0.1 M EDTA,
containing
0.5%
Brij 58, 0.2% deoxycholate and 0.5%
sodium lauroylsarcosine, pH 7.9, to which was then added
15 pl recombinant lysostaphin (Ambicin L; Aplin
&
Barrett)
solution (1 mg ml-' in 20 mM sodium acetate, pH 4.5) and
5
p1 chicken egg white lysozyme (Sigma) solution
(50
mg
ml-l in water). The treated cell suspension was vortexed and
a 300 pl aliquot was transferred to 300 pl warm (55 "C) 2%
low-gelling-temperature agarose (Seaplaque, FMC) pre-
pared with EC lysis buffer. The agarose/cell suspension was
immediately vortexed and then transferred to a well of a 10-
well sample plug mould (Bio-Rad). The agarose plug was
cooled for 10 min at room temperature. The cooled agarose
plug was removed from the mould with the aid of a small
spatula and then transferred to 3 ml EC lysis buffer and
incubated for 1-2 h at 37 "C until the plug was cleared
(complete lysis). Following lysis, the EC lysis buffer was
carefully decanted and replaced with 3 ml TE buffer (10 mM
Tris/HCl, 1 mM EDTA, pH 7.6). The agarose plug in TE
buffer was incubated at
55
"C for 1 h. The TE buffer was
decanted and replaced with 3 ml fresh TE buffer. The
agarose plugs were stored at 4 "C for up to 1 month.
Digestion of total DNA by the restriction endonuclease
SmaI
(for
Staphylococcus
species and
M.
caseolyticus,
which
have a DNA G
+
C content of less than 40 mol
%)
and
NotI
(for
M. equipercicus, M. bovicus, M. carouselicus,
B.
subtilis
and
Salinicoccus roseus,
which have a G+C content of
greater than 40 mol
YO)
was performed in a 1.5 ml micro-
centrifuge tube by using a 125 pl assay conducted according
to the instructions of the manufacturer (New England
Biolabs). A section (2 x4 x 1.5 mm) of the agarose plug
prepared as above was added to the assay tube and then
incubated with shaking (150 r.p.m.) for
2
h at 25 "C for
SmaI
assays or 37 "C for
NotI
assays.
PFGE procedures, macrorestriction pattern analysis and
genome size determinations were conducted as described
previously (George
&
Kloos, 1994).
Plasmid profile analysis.
The procedures for plasmid DNA
isolation and the agarose gel electrophoresis of the purified
plasmid preparations have been described previously
(DeGuglielmo
et al.,
1991), except that in the present study
the lysing buffer contained both lysostaphin
(500
pg ml-l)
and lysozyme (775 pg ml-l) as the staphylolytic enzymes and
electrophoresis was performed in horizontal agarose gels
(10 cm x 15 cm x 3 mm, 20 wells).
International Journal
of
Systematic Bacteriology
48
86
1
W.
E.
Kloos
and
others
Table
1,
List
of
Macrococcus
strains and their sources
Species
Strain designation
Source
DuPont
Kloos
ATCC
M.
caseolyticus
M.
bovicus
M.
equipercicus
M.
carouselicus
DD 4508
DD 4509
DD 4511
DD 4510
DD 4512
DD 4513
DD 7049
DD 7087
DD 7098
DD 6075
DD 4516
DD 4518
DD 4517
DD 9344
DD 9604
DD 9603
DD 9601
DD 9618
DD 9617
DD 9860
DD 9350
DD 9345
DD 9347
DD 9616
DD 9615
DD 9840
DD 9846
DD 9845
DD 11639
DD 11645
DD 1 1640
DD 11653
DD 11643
DD 11647
DD 11648
DD 11644
DD 11646
DD 11652
DD 11650
DD 11654
DD 11651
DD 11649
DD 9348
DD 9341
DD 9342
DD 9343
DD 9346
DD 9349
DD 9610
DD 9608
DD 9607
DD 9600
BT6
BL6
BH5
BHlO
GMWgl6
C2F4
C2F1
C2F24
H8a14
H9a16
H9b6
H9b7
H10a13
H10b15
H 17h3
H8h3
H8b6
H8bl2
H9b2
H9b10
H15b3
H17hl
H 17h2
H21al
H2 1 a9
H21h2
H21h3
H2 1 L4
H22h4
H22h5
H22h7
H23b2
H23 b6
H23b9
H23b10
H23bll
H23b12
H8b 16
H8al
H8a4
H8a10
H8b7
H8hl
H9a7
H9a10
H9b3
H9b9
1 3548T
29750
51834
51835
51825T
5 1826
5 1827
51831T
51832
5 1833
5
1
828T
5 1829
5 1830
Cow’s milk
Cow’s milk
Bovine tongue
Bovine lips
Bovine heart
Bovine heart
Food-processing factory
Food-processing factory
Food-processing factory
Pilot whale
Holstein cow
Holstein cow
Holstein cow
Irish thoroughbred horse
Morgan horse
Morgan horse
Morgan horse
Anglo-Trakehner horse
Anglo-Trakehner horse
Shetland pony
Irish thoroughbred horse
Irish thoroughbred horse
Irish thoroughbred horse
Morgan horse
Morgan horse
Shetland pony
Shetland pony
Shetland pony
Shetland pony
Shetland pony
Shetland pony
Shetland pony
Shetland pony
Shetland pony
Shetland pony
Shetland pony
Shetland pony
Shetland pony
Shetland pony
Shetland pony
Shetland pony
Shetland pony
Irish thoroughbred horse
Irish thoroughbred horse
Irish thoroughbred horse
Irish thoroughbred horse
Irish thoroughbred horse
Irish thoroughbred horse
Morgan horse
Morgan horse
Morgan horse
Morgan horse
862
International Journal
of
Systematic Bacteriology
48
Macrococcus
gen.
nov.
Table
7
(cont.)
Species
e
Strain
designation Source
DuPont
Kloos
ATCC
DD 9602
DD 9609
DD
9613
DD 9621
DD 9851
DD 9848
DD
9855
DD
11641
H9b14
H9h9
HlOalO
HlObl
H15b6
H15b14
H 16L2
H23b13
Morgan horse
Morgan horse
Anglo-Trakehner horse
Anglo-Trakehner horse
Shetland pony
Shetland pony
Shetland pony
Shetland pony
Cell
wall analysis.
Cell walls were purified with
4
%
SDS by
using the procedures of Glauner
et al.
(1988). The cell wall
peptidoglycan and teichoic acid analyses were performed as
described previously (Schleifer
&
Kandler, 1972
;
Anderson
et al.,
1977; Kaya
et al.,
1985; Webster
et al.,
1994).
Phosphate levels were determined by the method
of
Ames
(1966). Lipoteichoic acid analyses were performed as de-
scribed for
Staphylococcus
species (Ruhland
&
Fiedler,
1990).
DNA
base composition.
The DNA G+C content was
determined by the thermal denaturation method of Marmur
&
Doty (1962) using a Gilford Response Spectrophotometer
with Response
I1
Thermal Programming (Gilford Systems).
RESULTS
AND
DISCUSSION
Phylogenetic position and analysis
of
16s
rRNA
sequences
Phylogenetically,
Staphylococcus caseolyticus
(Schleifer
et al.
1982) and the related group of species
previously designated as
'
Staphylococcus equiper-
cicus
',
'
Staphylococcus bovicus
'
and
'
Staphylococcus
carouselicus'
(Ballard
et al.
1995) were separated from
other recognized staphylococci to such an extent that
they should be considered as members of a separate
genus, herein designated
Macrococcus
gen. nov. The
intergroup 16s rRNA sequence similarities were 93.4-
95.3
YO,
suggesting that macrococci represent the sister
group of the staphylococci. The monophyletic status
of both groups is well-documented in the unrooted tree
of Fig. 1 and by overall intragroup 16s rRNA sequence
similarities of
2
97.7
YO
for macrococci and
2
96.5
YO
for staphylococci. The sequence similarities of these
two groups and
B.
subtilis
and
Salinicoccus roseus,
their next shown relatives among the Gram-positive
bacteria with a low DNA G+C content, were 90.5-
92-9
%.
The data shown in Fig. 1 are based on at least
90
%
complete (in comparison with the 16s rRNA
from
E.
coli)
16s rRNA primary structures from each
of
the representative species. All other staphylococcal
species not shown, but yet represented by complete or
partial 16s rRNA sequence data in public (Maidak
et
al.,
1996; Van de Peer
et al.,
1996) and other databases
clearly clustered among the staphylococci group, i.e.
Staphylococcus arlettae, Staphylococcus caprae,
Staphylococcus cohnii, Staphylococcus equorum,
Staphylococcus hyicus, Staphylococcus kloosii,
Staphylococcus lugdunensis, Staphylococcus pisciferm-
entans
and
Staphylococcus xylosus.
DNA-DNA hybridization and DNA base compostion
The DNA relationships and DNA G+C content of
selected
Macrococcus, Staphylococcus, Salinicoccus
and
B.
subtilis
strains are shown in Table 2. Each of the
Macrococcus
species was represented by three strains,
one
of
which was the type strain. The
Staphylococcus
species selected were those found living together with
Macrococcus
species
on
the same host species (i.e.
artiodactyls, perissodactyls and cetaceans), and the
more distantly related species
Staphylococcus epider-
midis
that is occasionally found as transient popu-
lations on these hosts. In addition,
Staphylococcus
auricularis
was selected for this study because it has
been reported (Schleifer, 1986) that this species has an
unusual teichoic acid like that proposed for
Staphylo-
coccus caseoly ticus,
i. e. a
poly(Wacetylglucosaminy1-
phosphate) type. As can be seen from the data, each
proposed
Macrococcus
species formed a separate, well-
defined DNA similarity group under optimal
(55
"C)
and stringent (70 "C) conditions. Strains of the same
Macrococcus
species demonstrated a relatively high
level of DNA relatedness (mean relative binding of
78 6
%
at
55
"C and 72
f
3
%
at 70
"C,
shown as bold
numbers in the table), whereas strains of different
Macrococcus
species demonstrated a lower level
of
DNA relatedness (mean relative binding of 33 12
%
at
55
"C and 14+
3
YO
at 70 "C). The species
M.
equipercicus, M. bovicus
and
M. carouselicus
were
more closely related to one another than to
M.
caseolyticus
and formed a species group (Kloos, 1980;
Kloos
&
Schleifer, 1981), which herein is informally
International Journal
of
Systematic Bacteriology
48
863
W.
E.
Kloos
and others
St.
aureus
ATCC
1
2600T
b
St.
muscae
CCM
4175T
St.
carnosus
DSM
20501T
.
..............
....
...................................................................
.
......
........
St.
auricularis
ATCC
33753'
Fig.
1,
165
rRNA tree reflecting the
phylogenetic relationships
of
the type
strains of
Macrococcus
species and a
representative selection of
Staphylococcus
(St.)
species, as well as
Salinicoccus (Sa.)
roseus
and
B.
subtilis.
The latter two
organisms indicate the level of relatedness
of the
Ma croco ccus-Sta phylococcus
branch
and the other Gram-positive bacteria with a
low DNA G
+
C
content. The tree
is
based on
the results of a maximum-likelihood
analysis. The tree topology was corrected
according to the data obtained by applying
distance and maximum-parsimony methods.
Only sequence positions which shared
identical nucleotides in at least
50%
of
all
available sequences
of
macrococci and
staphylococci were used to construct the
tree. Bar,
5
substitutions per
100
nucleotides.
5%
Sa. roseus
ATCC
49258T
designated the
Macrococcus equipercicus
species
group. The DNA relatedness of
Macrococcus
species
to members of the
Staphylococcus sciuri
species group
(Staphylococcus sciuri, Staphylococcus lentus
and
Staphylococcus vitulus)
appeared to be slightly higher
than to the other staphylococcal species (14f3 vs
11
f
2% at 55 "C). Furthermore, of the four macro-
coccal species,
M. caseolyticus
appeared to be the most
closely related to the
Staphylococcus sciuri
species
group (19
f
0.5 vs 13
&
1
%
at 55
"C).
There was a good
correlation between the results of DNA-DNA hybrid-
ization and the analysis of 16s rRNA sequences (Fig.
1) in that both methods suggested the genus
Macro-
coccus
is more closely related to
Staphylococcus sciuri
than to staphylococcal species outside the
Staphylo-
coccus sciuri
species group.
The DNA
G
+
C content of 41-45 mol
YO
for members
of the
M. equipercicus
species group is higher than that
of recognized
Staphylococcus
species (Kloos
&
Schleifer, 1986
;
Kloos
et al.,
199 1
;
Kloos, 1998) (Table
2) and these results further support the inclusion of
these species in a separate genus. The DNA G+C
content of 38-39 mol% reported for M.
caseolyticus
(Schleifer
et al.,
1982) was shared by several of the
staphylococcal species and this feature is consistent
with the results of DNA-DNA hybridization, suggest-
ing that this species is more closely related to staphylo-
cocci than are members
of
the M.
equipercicus
species
group.
Normalized ribotype patterns
An ensemble of ribotype pattern types sharing com-
mon features and not fitting into any known species
structure potentially constitutes an undescribed spe-
cies. This statement is dependent on knowledge of the
polymorphic pattern structures surrounding the newly
recognized species. With this in mind, we determined
the ribotype patterns of approximately 2500 strains
representing 3 1 species of
Staphylococcus
(Hubner
et
al.,
1993; Cole
et al.,
1994; Webster
et al.,
1994) and
compared them with patterns of strains representing
the new
Macrococcus
species and M.
caseolyticus
(Tables
1,
2). When sorted by similarity analysis,
ribotype pattern groups representing strains
of
M.
equipercicus,
M.
bovicus,
M.
carouselicus
and
M.
caseolyticus
appeared closer to one another than to
any of the
Staphylococcus
species. Each of the above
four species representations (Fig. 2) had a unique
combination of conserved fragment sizes detected by
hybridization with the labelled rRNA operon probe.
All of the pattern types of
M.
caseolyticus
contained
bands at 0.76, 1-05, 1-3, 2-3 and 3.9 kbp; all
M.
equipercicus
patterns contained bands at 1.1, 2.3, 2.6,
4-2 (except dd 11648) and 10 kbp; all
M.
bovicus
patterns contained bands at
1.1
(except dd 09617), 1.3,
2, 2.4 (except dd 04518) and 4 (except dd 09344) kbp;
and all
M.
carouselicus
patterns contained bands at
1.05, 1.3, 1.7, 2.4 and
8
kbp. In support of the transfer
of
Staphylococcus caseolyticus
to the genus
Macro-
coccus,
all of the above four species demonstrated
conserved fragments of 1.05-1-1 kbp and 9.5-10 kbp,
whereas some
Staphylococcus
species demonstrated a
slightly smaller conserved fragment of
1-0
kbp and
only occasionally had a fragment of about 10 kbp (Fig.
2). Furthermore, several of the lower
Staphylococcus
species, such as
Staphylococcus simulans, Staphylo-
coccus carnosus, Staphylococcus schleiferi, Staphylo-
864
International Journal
of
Systematic Bacteriology
48
Macrococcus
gen. nov.
Table
2.
Results of hybridization
of
Macrococcus,
Staphylococcus,
Salinicoccus
and
Bacillus
DNA
with
[rnethyl-
3H]thymidine-labelled
DNA
from each of the
Macrococcus
species under optimal
(55
"C)
and stringent
(70
"C)
conditions
Genus and Strain DNA G+C
species
(mol
%)
M.
caseolyticus
M.
equipercicus
M.
bovicus
M.
carouselicus
Staphylococcus
vitulus
Staphylococcus
lentus
Staphylococcus
sciuri
Staphylococcus
muscae
Staphylococcus
hyicus
Staphylococcus
chromogenes
Staphylococcus
gallinarum
Staphylococcus
equorum
Staphylococcus
kloosii
Staphylococcus
auricularis
Staphylococcus
aureus
Staphylococcus
epiderm idis
Salinicoccus
roseus
B.
subtilis
DD 4508T
DD
4511
DD 6075
DD 9350T
DD 9615
DD 9840
DD 4516T
DD 4517
DD 9344
DD
9348T
DD 9341
DD 9607
DD 756T
DD 4203T
DD
4277T
DD 4795T
DD 887T
DD 851T
DD
854T
DD 853T
DD 857T
DD 872T
DD
10156T
DD 8726T
DD 12047T
DD
379T
38
ND
ND
45
45
44
42
41
41
34
34
37
40
33
34
35
35
36
39
33
34
49
43
ND
ND
ND
Percentage relative binding with labelled DNA
from
:
M.
caseolyticus
M.
equipercicus
M.
bovicus
M.
carouselicus
DD
4508T DD 9350T
DD
4516T DD 9348T
55°C 70°C 55°C 70°C 55°C 70°C 55°C 70°C
100
74
73
25
27
26
30
24
22
25
25
23
19
19
18
13
15
12
12
16
17
11
14
12
11
10
100
69
73
14
16
17
17
15
13
11
10
9
9
5
8
5
4
6
7
8
6
ND
ND
ND
ND
ND
28
29
24
100
80
71
32
37
34
47
37
37
13
12
12
11
12
9
12
11
10
9
10
ND
12
10
10
10
16
100
72
69
17
14
11
21
12
8
7
7
1 7.
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
21
24
21
40
44
42
100
80
72
58
50
54
13
11
12
14
11
11
10
10
10
9
9
9
10
9
11
9
10
15
14
17
100
68
76
18
16
9
6
9
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
17
18
18
36
41
28
51
54
47
100
89
81
15
12
13
11
10
9
14
9
12
10
13
12
10
9
13
9
10
18
17
17
15
16
100
74
74
8
9
7
ND
ND
ND
ND
6
ND
8
ND
9
7
ND
ND
ND,
Not determined.
coccus delphini, Staphylococcus chromogenes, Staphyl-
ococcus lentus, Staphylococcus vitulus
and
Staphyl-
ococcus sciuri,
did not even have a fragment in the size
range of 0.8-1.3 kbp. Each of the
DNA
similarity
groups examined in Table
2
manifested a different
ribotype pattern.
Macrorestriction pattern analysis and estimation
of
genome size
The restriction endonuclease
SmaI
(recognition site:
5'
.
. .
CCCGGG
.
.
.3')
is commonly used to digest the
chromosomes of staphylococci for macrorestriction
~~ ~ ~~
International Journal
of
Systematic Bacteriology
48
865
W.
E.
Kloos
and others
Fig.
2.
Diversity of normalized ribotype pattern types found within each of the
Macrococcus
species and
a
representative
ribotype pattern type of selected species of the related genera
Staphylococcus
(St.)
Salinicoccus (Sa.)
and
Bacillus.
Certain
Macrococcus
pattern types are likely to represent many strains, as shown by the strain count.
pattern analysis (Lina
et
al.,
1992; George
&
Kloos,
1994; Bannerman
et
aZ.,
1995). It cleaves the
Staphy-
Zococcus
chromosome into about
7-30
fragments,
depending upon the particular species and strain. This
enzyme cleaved the chromosomes
of
M.
caseolyticus
strains
into
29-34 fragments, most
of
which were well
866
In terna ti0 na l
lo
urna l
of
Systematic Bacteriology
48
Macrococcus gen. nov.
Fig.
3.
Macrorestriction pattern analysis of Srnal-digest fragments of chromosomes
of
M.
caseolyticus (a), and Notl-digest
fragments of chromosomes
of
M.
carouselicus (b),
M.
equipercicus (c) and
M.
bovicus
(d), separated by
PFGE.
separated following PFGE (Fig. 3). The ten strains
produced eight different macrorestriction patterns.
Three strains
(DD
7087,
DD
7049 and
DD
7098)
isolated from the same food-processing factory pro-
duced a very similar pattern. These strains could be
distinguished only on the basis of their plasmid profile
(data not shown) and they probably represent rela-
tively recent subclones. SmaI-digest fragments of sizes
2.9,3.0,3.2,9-5,23,40,45,68,99
and 108 kb are highly
conserved among
M.
caseolyticus strains. Based on the
sum of the sizes of SmaI-digest fragments, the esti-
mated size of the M. caseolyticus genome is 1714k
93 kb (Table 3).
SmaI cleaved the chromosomes of members of the M.
equipercicus species group into a very large number
of
small fragments, many of which were overlapping and
not clearly distinguishable by PFGE. Several other
restriction endonucleases that recognize GC hexa-
nucleotide sequences (NaeI, NarI and SacII) and GC
octanucleotide sequences (NotI, FseI and AscI) were
tested for their ability to produce definable macro-
restriction patterns. Of these enzymes, the restriction
endonuclease NotI (recognition site
:
5’
.
.
. GCGGC-
CGC
.
. .3’) was most satisfactory for members of the
M. equipercicus species group in that it cleaved their
chromosome into a relatively small number of frag-
ments, most of which were well-separated by PFGE.
This enzyme cleaved the chromosomes of 21 of the 22
strains of M. equipercicus into 33-37 fragments (Fig.
3). These strains produced 19 different macro-
restriction patterns. The chromosome of strain
DD
9347 appeared to be only partially cleaved at two sites
by NotI, as indicated by the presence of a single faint
band migrating in the gel. It is quite possible that some
strains of macrococci produce site-specific
DNA
International Journal
of
Systematic Bacteriology
48
867
W.
E.
Kloos and others
Table
3.
Comparison
of
the genome sizes
of
Macrococcus species with those
of
Staphylococcus
and
Salinicoccus species
Genus and
species
No.
of
Genome fragments Estimated
strains produced
by
:
genome size
(kb)
M.
caseolyticus
M.
equipercicus
M.
bovicus
M.
carouselicus
Staphylococcus
aureus
Staphy lococcus
epiderm idis
Staphylococcus
capitis
Staphylococcus
caprae
Staphylococcus
hominis
Staphylococcus
schleiferi
Staphylococcus
sciuri
Salinicoccus
roseus
10
20
8
16
26
13
26
13
26
10
9
1
SmaI
NotI
NotI
NotI
SmaI
SmaI
SmaI
SmaI
SmaI
SmaI
SmaI
NotI
1714f93
1536
f
91
1753
f
64
1706f95
2741
f
232
2365
f
124
2365
f
122
2584f 146
2372
f
137
2283
f
171
2697
f
162
204 1
methylases inhibiting digestion by NotI, for this
enzyme is sensitive to certain types of site-specific
methylation. Many of the NotI-digest fragments of M.
equipercicus strains were highly conserved, especially
those of sizes
2,3.2-3.8,4.2,6.5,7,8,83,
10, 12, 14, 15,
16, 18, 22, 25, 28, 34, 36, 48, 57, 79 and
88
kb. The
estimated size of the
M.
equipercicus genome is
1536
_+
9
1
kb (Table
3),
though it may be slightly larger
since the number of discrete fragments present in
several of the intensely stained bands is uncertain.
NotI cleaved the chromosomes of nine of the ten
strains of M. bovicus into 14-17 well-separated frag-
ments (Fig. 3). The nine strains produced eight
different macrorestriction patterns. The chromosome
of
strain DD 9603 appeared to be only partially
cleaved at two sites by NotI, as indicated by the
presence of a single faint band following PFGE. NotI-
digest fragments of sizes 4.9, 12, 19, 28, 44 and 97 kb
were highly conserved among
M.
bovicus strains. The
estimated size of the
M.
bovicus chromosome is
1753
f
64 kb (Table 3).
The chromosomes of 16 of the 17 strains of M.
carouselicus were cleaved by NotI into 12-16 well-
separated fragments (Fig. 3). The 16 strains produced
15 different macrorestriction patterns. The chromo-
some of strain DD 9608 appeared to be cleaved by
NotI at only two sites, as indicated by the presence of
a single, well-defined band following PFGE. NotI-
digest fragments of sizes 6.9, 11, 16, 26, 39,
56
and
100 kb were highly conserved among
M.
carouselicus
strains. The estimated size of the
M.
carouselicus
chromosome is 1706
&
95 kb (Table
3).
As
can be seen
from the data in Table 3, the genome sizes of
Macrococcus species are significantly smaller than
those of the Staphylococcus species for which there
have been calculations of genome size.
Cell
wall
composition
The cell-wall peptidoglycan and teichoic acid com-
position were determined for several strains of each of
the Macrococcus species, including
M.
caseolyticus
DD 4508T and DD 4509 determined previously
(Schleifer
et
al., 1982),
DD
4510 and DD 6075;
M.
equipercicus DD 9350T, DD 9615 and DD 9840; M.
bovicus DD 4516T, DD
4518
and DD 9344; and
M.
carouselicus
DD
934gT, DD 9341 and DD 9607. The
peptidoglycan type for macrococci is ~-Lys-Gly,-,,
L-
Ser, a basic type that is also found among several
staphylococcal species (Schleifer, 1986
;
Kloos, 1998).
Some differences were noted in the levels
of
L-serine in
the peptidoglycans of certain macrococcal species, for
example,
M.
caseolyticus strains exhibited the highest
levels [1-2-1.3 mol (mol glutamic acid)-'] and
M.
bovicus exhibited the lowest levels
[0-44-0.58
mol (mol
glutamic acid)-l]. The cell wall teichoic acid type of
M.
caseolyticus strain DD 4508T has been reported to be
poly( N-acet ylgalact osamin ylp hosp hate), where the
N-
acetylaminosugar residues form an integral part of the
868
International Journal
of
Systematic Bacteriology
48
Macrococcus
gen. nov.
. . . . . .
.
. . . . . .
.
.
.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.
. . .
.
. .
.
. .
.
.
,
.
. .
.
.
.
,
. . . .
,
.
,
. . . . . .
.
. .
,
. . . . . .
, ,
. . .
,
. .
,
.
,
.
, ,
. . .
,
.
,
. .
,
.
,
.
,
,
. .
.
. . . . .
, ,
. . .
,
. .
,
. . . . .
, ,
. .
.
. .
.
.
. . . .
.
. . . . . . . . . . .
.
. . . . . .
. .
. . . . .
. .
. . . . . . . .
.
,
. . .
,
. . . . . . . . .
,
. .
,
.
.
.
.
. .
.
.
.
. . . .
.
. . . . . . . . . .
.
. . . . . . . . .
.
. . . .
,
.
. .
.
. .
. . . . .
.
.
. . .
. .
. . . . . .
.
. . .
. .
. . . . .
.
. . . . . . . . . . .
.
. . .
.
.
.
. . .
.
Fig.
4.
Phase-contrast photomicrographs
of
Gram-stained cells
of
(a) the type strain
of
M.
carouselicus,
(b) the type strain
of
Staphylococcus sciuri
subsp.
sciuri,
(c)
a
mixture of the type strains of
M.
carouselicus
(larger 'glowing' cells in clusters)
and
Staphylococcus aureus
(small blue-outlined cells occurring singly and in pairs, small clusters and short chains), and (d)
a
mixture
of
the type strains
of
M.
carouselicus
(larger 'glowing' cells in clusters) and
Staphylococcus sciuri
subsp.
sciuri
(smaller 'glowing' cells occurring singly and in pairs and small clusters). Cells were grown in
TS
broth for
18
h
at
35
"C.
Bar,
4
pm.
polymer chain (Schleifer, 1986). However, since only
very low amounts of phosphorus
[Om45
pmol (mg cell
wall)-l] and glycerol could be detected in the cell wall
upon reinvestigation of the type strain, it appears that
any teichoic acid present in M.
caseolyticus
would be in
an amount much lower than that found in the cell walls
of staphylococci. The teichoic acid of
M.
caseolyticus
strain
DD
6075 also contained N-acetylgalactosamine,
whereas strain
DD
4509
contained N-acetylglucos-
amine as the N-acetylaminosugar residue. The cell
walls
of
members
of
the M.
equipercicus
species group
did not contain detectable levels of teichoic acids, as
indicated by the absence of both phosphorus and
polyol residues. By contrast, the cell walls of all
currently recognized
Staphylococcus
species contain
teichoic acids (End1
et al.,
1983
;
Schleifer, 1986; Kloos,
1998). As
for
the genus
Staphylococcus
(Ruhland
&
Fiedler, 1990), lipoteichoic acids were found to be
present at the cell surface of the type strains of M.
caseolyticus,
M.
equipercicus,
M.
bovicus
and M.
carouselicus.
The glycosyl residue of the glycolipid is
gentiobiosyl, which is the same glycosyl as that found
in staphylococci. The fatty acid substitution was not
investigated.
Phenotypic characterization
Cells of macrococci were Gram-positive and 1.1-
2.5
pm in diameter, depending on the species and
growth medium. They were non-motile and non-spore-
forming, and were arranged mostly in pairs and
tetrads, and occasionally single or arranged in short
chains. Cells of
M.
equipercicus
and
M.
carouselicus
were usually larger than those of the other macrococcal
species. When grown in TS broth, the Gram-stained
cells of macrococci were approximately
2-5-4
times the
International Journal
of
Systematic Bacteriology
48
869
W.
E.
Kloos and others
fig.
5.
Scanning electron micrographs
of
the type strains
of
(a)
M.
caseolyticus,
(b)
M,
equipercicus, (c)
M.
bovicus
and
(d)
M.
carouselicus. Magnification
x
13
320.
diameter of
Staphylococcus aureus
cells and
1-5-2.5
times the diameter of
Staphylococcus sciuri
cells. The
difference in cell size between the two genera was less
remarkable when cells were grown in
BHI
broth or on
TSA, TSA plus
5
%
sheep blood,
BHI
agar or
P
agar.
Cells of
M.
equipercicus
were arranged predominantly
in tetrads. We found serendipitously that Gram-
stained cells
of
macrococci, when viewed under the
phase-contrast microscope, appeared much larger than
staphylococci and their cells reflected a glowing pink-
or orange-brown colour (Fig. 4). Scanning electron
microscopy (SEM) revealed that the surface of
M.
caseolyticus
cells was quite smooth, whereas the
surface of
M.
bovicus
and M.
carouselicus
cells was
somewhat irregular, and numerous small piliform
projections were present on the surface of M.
equi-
percicus
cells (Fig.
5).
Macrococci are marginally facultative anaerobes.
Growth was much better under aerobic conditions
than under anaerobic conditions. Members of the
M.
equipercicus
species group do not grow significantly in
the anaerobic portion of a thioglycollate semi-solid
medium; however, some strains of
M.
caseolyticus
grow slowly in the anaerobic portion of this medium.
Macrococci were positive for catalase and oxidase
(Microdase disk test) activities, resistant to the 0.04 U
bacitracin (Taxo A) disk and lysozyme
(25
pg ml-l),
and susceptible to the 100 pg furazolidone disk. They
were negative for staphylocoagulase, alkaline phos-
phatase, ornithine decarboxylase, P-glucuronidase and
P-galactosidase activities and arginine utilization, and
did not produce acid from D-mannose, D-melezitose,
D-xylose, L-arabinose, D-turanose, D-sorbitol, D-cello-
biose, salicin and D-raffinose. Members of the
M.
equipercicus
species group were susceptible to lyso-
staphin
(10
pg applied as a drop to the inoculated agar
surface), whereas M.
caseolyticus
was slightly resistant
to this enzyme.
Macrococci were susceptible to a wide range of
antibiotics and did not exhibit the resistance profiles
characteristic of most staphylococcal species. Using
the breakpoints established for disk-diffusion testing
of
Staphylococcus aureus,
all macrococcal strains
tested were susceptible to penicillin
G,
erythromycin,
clindamycin, tetracycline, ciprofloxacin, rifampin, tri-
met hoprim-sul famethoxazole, gent amicin, kanamy
-
cin, streptomycin, vancomycin and chloramphenicol.
M.
equipercicus
strain
DD
11653 was resistant to
870
International Journal
of
Systematic Bacteriology
48
Macrococcus
gen.
nov.
oxacillin, and strains DD 11644, DD 11648 and DD
11 649 showed intermediate resistance to oxacillin; all
other macrococci examined in this study were sus-
ceptible to this antibiotic. The mechanism for oxacillin
resistance in these organisms is unknown. The gene
mecA
appeared to be absent in the above strains since
amplification products were not detected in PCR using
a set of primers capable of detecting
mecA
homologues
in staphylococci (Kloos
et al.,
1998). M.
equipercicus
strain DD 11651 and M.
bovicus
strain DD 9860 were
resistant to lincomycin. Members of the M.
equi-
percicus
species group were resistant to novobiocin
(5 pg, disk inhibition zone diameters of 12-1 6 mm), on
the basis of the breakpoint of 16 mm established for
Staphylococcus saprophyticus
(Kloos
&
Bannerman,
1995). M.
caseolyticus
strains exhibited inhibition zone
diameters of 12-1
8
mm with novobiocin disks, classi-
fying them as either resistant or susceptible.
Characters that were variable in one or more of the
Macrococcus
species are listed in Table 4. Several of
the phenotypic characters were expressed differently
among the species and therefore had taxonomic value.
For example, acetoin was produced by all of the strains
of M.
caseolyticus,
but was not detected in the other
species. M.
caseolyticus
reduced nitrates, whereas M.
equipercicus
and all but one strain of M.
bovicus
and
M.
carouselicus
failed to reduce nitrates. M.
carou-
selicus
exhibited a moderate to strong DNase activity.
Only one strain of
M.
equipercicus
had detectable
DNase activity, and this was weak. Most strains (86
%)
of
M.
equipercicus
exhibited moderate to strong urease
activity, whereas none of the
M.
caseolyticus
and
M.
carouselicus
strains and only two of the M.
bovicus
strains had detectable urease activity. Most strains
(80
YO)
of M.
caseolyticus
exhibited pyrrolidonylaryl-
amidase activity. Members of the M.
equipercicus
group did not exhibit this enzyme activity. M.
bovicus
and most strains (95%) of
M.
equipercicus
produced
acid aerobically from D-mannitol, but
M.
caseolyticus
failed to produce acid from this carbohydydrate. Most
strains (94%) of M.
carouselicus
produced only low
levels of acid from P-D-fructose, whereas the other
species produced noticeably higher levels of acid from
this carbohydrate.
M.
caseolyticus
produced acid from
maltose, but
M.
carouselicus
did not produce acid
from this carbohydrate.
M.
bovicus
and most strains of
M.
caseolyticus
(90
%)
demonstrated a partial hae-
molysis (greening) of horse blood in
TSA
following
24-48 h incubation at 35 "C. Haemolysis was not
observed with M.
equipercicus
and most strains (94
YO)
of M.
carouselicus.
On the basis of phenotypic charac-
ters,
M.
caseolyticus
is the most distinguishable of the
macrococcal species. The lower phenotypic similarity
was supported by the genotypic data separating this
species from the M.
equipercicus
species group.
Plasmid profiles
An examination of plasmid carriage was prompted by
the results of antibiograms showing the absence among
macrococci
of
antibiotic resistances (e.g. to penicillin
G, erythromycin, clindamycin, tetracycline, genta-
micin, kanamycin, streptomycin and chlorampenicol)
commonly determined by plasmid-borne genes in most
staphylococcal species and other related bacteria
(Lyon
&
Skurray, 1987; Novick, 1989; Archer
&
Climo, 1994; Kloos, 1998). Consequently, we con-
sidered the possibility that macrococci either do not
carry plasmids or carry them infrequently. However,
most of the macrococci carried one or more cryptic
plasmids, including all of the
M.
caseolyticus
strains,
95
YO
of M.
equipercicus
strains, 94
YO
of
M.
carousel-
icus
strains and 70% of M.
bovicus
strains. The
macrococci demonstrated a mean number of 4&3
different plasmids per strain (based on the recognition
of plasmid bands suspected of representing covalently
closed circular forms). Most strains (86%) of M.
equipercicus
had 3 and
5
kb plasmids that produced a
noticeable series of structural forms, i.e. a multi-form
ladder, on agarose gels. Several strains of the other
macrococcal species had plasmids of these same sizes.
Description
of
Macrococcus
gen. nov.
Macrococcus
(Ma.cro.coc'cus. Gr. adj.
macrus
large
;
Gr. masc. n.
kokkos
a grain or berry; M.L. masc. n.
Macrococcus
a large coccus).
The description below is based on data collected on the
species
Staphylococcus caseolyticus
(Schlei fer
et al.,
1982; End1
et
al.,
1983; Schleifer, 1986) and data from
this study. Cells are spherical or coccoid, Gram-
positive, non-motile, non-encapsulated and do not
produce endospores. They are 1-1-2.5 pm in diameter
and are larger than the cells of
Staphylococcus
species
when comparisons are made of cells grown on the
same medium. Metabolism is mainly respiratory;
growth is chemoorganotrophic and only marginally
facultatively anaerobic. They are catalase- and oxi-
dase-positive, staphylocoagulase-, ornithine-de-
carboxylase-, alkaline-phosphatase-, P-glucuronidase-
and P-galac tosidase-nega tive, non- halop hilic and
mesophilic. They are resistant to bacitracin and
lysozyme, and susceptible to furazolidone. The pep-
tidoglycan type is ~-Lys-Gly,-,, L-Ser, and only one of
the four macrococcal species, i.e.
Macrococcus caseo-
lyticus,
appears to contain cell-wall teichoic acid.
Lipoteichoic acid is present. The genome size range is
approximately 150G1800 kb. The
G
+
C content of
the DNA is 38-45 mol
YO
[as determined by the thermal
denaturation method (Marmur
&
Doty, 1962)l. The
type species is
Macrococcus equipercicus.
It was selec-
ted as the type species in place of M.
caseolyticus,
the
first described species of the group, because it is the
most typical of the cluster of four species now
recognized.
Description of Macrococcus caseoryticus (Schleifer et
a/.
1982)
comb. nov.
Macrococcus caseolyticus
(ca.se.o.1y'ti.cus. L. n.
caseus
cheese; Gr. adj.
lyticus
able to loose; M.L. adj.
caseolyticus
casein-dissolving).
International Journal
of
Svstematic Bacterioloav
48
W.
E.
Kloos
and others
Table
4.
Variable characteristics
of
Macrococcus
species
Characteristic Percentage
of
strains positive (and weakly positive)
M.
caseolyticus
M.
equbercicus
M.
bovicus
M.
carouselicus
(n
=
10)
(n
=
22)
(n
=
10)
(n
=
18)
P
agar colony
diameter
2
4
mm
TSA colony
diameter
3
6 mm
Anaerobic growth
Colony pigmentation
:
orange
Colony lustre
:
glistening
Haemoly sis
(horse blood)
Acetoin production
Aesculin hydrolysis
Nitrate reduction
DNase
Urease
Pyrrolidonylarylamidase
P-Glucosidase
Staph Latex
agglutination
Lysostaphin
resistance*
Novobiocin
resistance?
Oxacillin resistance$.
Acid (aerobic)
D-Mannitol
Maltose
Sucrose
Glycerol
D-Ri bose
a-Lactose
P-D-Fructose
D-Trehalose
produced from
:
20
80
0
(50)
0
100
0
(90)
100
0
(20)
100
40
0
80
0
90
50
(50)
80
(20)
0
0
100
40
90
80
60
100
100
95
95
0
95 (5)
23
0
0
0
86
0
45
(23)
0
(5)
0
(5)
0
(5)
0
100
5 (14)
95
32
(41)
0 (9)
100
0
(5)
0
100
32
(18)
80
20
0
70
100
0
(100)
0
10
10
20
0
50
30
30
(10)
0
(10)
100
0
100
50
(20)
0
(20)
100
10
0
100
50
(10)
83
94
0
0
(67)
100
0
(6)
0
94
6
100
0
0
6
33
0
100
0
6
(33)
0
67
0
0
0
(50)
0
(94)
61
(33)
*
Growth response on the surface of a
TSA
plate in the presence of a 0.05 ml drop
of
distilled water
containing 10 pg lysostaphin. Positive, resistant, confluent growth; weakly positive, intermediate
resistance, reduction in growth in the area
of
the drop; negative, susceptible, no growth.
t
Disk containing
5
pg; positive, resistant, zone ofinhibition
2
16 mm; weakly positive, intermediate
resistance, zone of inhibition 17-18 mm.
$
Disk containing 1 pg; positive, resistant, zone of inhibition
3
10 mm; weakly positive, intermediate
resistance, zone of inhibition 11-12 mm.
The description below is based on characteristics
previously described (Schleifer
et
al.,
1982) and this
paper.
A
total of ten strains were examined (Table I),
including strains
DD
4508T
(=
ATCC 13548T) and
DD
4509
(=
ATCC 29750) isolated from cow's milk,
and an additional eight strains isolated in 1992 from
the skin of a whale and from raw beef and other meat
products. Colonies grow to 3
f
1
mm in diameter on
P
agar and 7
f
1
mm in diameter on TSA. They are
slightly convex, entire, butyrous, glistening and
opaque, and may be unpigmented (grey-white) or have
a pale yellow pigmentation. Growth in the anaerobic
portion
of
a semi-solid thioglycollate medium ranges
from no detectable growth to weak, delayed growth.
A
small amount of L-(+)-lactic acid is produced from
glucose under anaerobic conditions. Growth is good at
NaCl concentrations up to 10%. Optimum growth
temperature is
35
"C. The cytochromes of strains
DD
872
International Journal
of
Systematic Bacteriology
48
Macrococcus
gen. nov.
4508T and DD 4509 are
aa,,
c,~~,
c,,,,
b,,,
and
b,,,,
and
aa,,
c,~~,
c,,~,
b,,,
and
b564,
respectively. Both of these
strains produce a class I1 fructose- 1,6-diphosphate
aldolase. The cell wall contains only small amounts of
phosphorus and glycerol and appears to have an
atypical teichoic acid of the type poly(N-acetylgalacto-
saminylphosphate) [or
poly(N-acetylglucosaminyl-
phosphate)]. Acetoin is produced and nitrates are
reduced. Acid is produced aerobically from maltose,
#?-
D-fructose and D-trehalose. Acid is not produced from
D-mannitol. Urease and #?-glucosidase activities are
negative. Characteristics of
M. caseolyticus
that are
general properties of the genus are listed above. The
variable characteristics for this species are listed in
Table 4. The API STAPH-IDENT profile is 0400.
Major
ID32
STAPH profiles are 0661 30300 (30
YO),
062130300 (30%) and 066130101 (30%). The G+C
content of the DNA is 38-39 mol
YO.
This relatively
uncommon species appears to have a preference for
artiodactyl (e.g. cattle, sheep and goats) and cetacean
(e.g. whale) hosts and may be found in their milk and
meat products.
Amended
description
of
the
type
strain. Type strain is
ATCC 13548T (Schleifer
et al.
1982)
(=
DD 450ST),
isolated from cow's milk, which was previously desig-
nated the type strain of
Staphylococcus caseolyticus
(Schleifer
et al.
1982). It has all of the general properties
of the species and genus described above and also the
following characteristics. Cells are spherical, 1.1-2 pm
in diameter, have a smooth surface (SEM), and occur
singly, in pairs, short chains and clusters. Colonies on
P agar and TSA are circular,
4-5
mm in diameter,
entire, low convex, and possess a smooth and glistening
surface and a sticky consistency. They are grey-white
with slight cream-yellow pigmentation near the colony
edge. Cells are facultatively anaerobic and show
delayed weak growth in the anaerobic portion of
thioglycollate medium. No haemolysis of sheep, horse
and bovine blood. Moderate pyrrolidonylarylamidase
activity. Positive latex agglutination.
No
DNase
activity and aesculin hydrolysis. Acid produced
aerobically from D-glucose,
P-D-
fruct
o
se, a-lact
o
se,
D-ribose, D-galactose, D-trehalose and maltose.
No
acid produced from D-mannitol, sucrose and glycerol.
Susceptible to marginally resistant to novobiocin.
Slightly resistant to lysostaphin. Type dd 04508 ribo-
type pattern. Class
I1
fructose- 1,6-diphosphate aldo-
lase. Cytochromes
aa,,
b
and
c
are present. Cell wall
peptidoglycan L-LYs-G~Y,, L-Ser. The presumed tei-
choic acid contains glucose and N-acetylgalactosamine
and only small amounts of N-acetylglucosamine and
glycerol. Lipoteichoic acid is present.
G
+
C content of
DNA
is
38 mol
YO.
Description
of
Macrococcus
equipercicus sp. nov.
Macrococcus equipercicus
(equi.per'ci.cus.
L.
gen. n.
equi
of a horse. M.L. adj.
equipercicus
pertaining to a
horse named Percy, from which this species was first
isolated).
The description below is based on the characteristics of
22 strains (Table 1) isolated in 1993-1995 from the skin
of horses and ponies. Colonies grow to 6+2 mm in
diameter on P agar and 6+
1
mm
on
TSA. They are
convex, entire, butyrous, dull to slightly glistening and
opaque, and have a light- to medium-orange pig-
mentation. Growth is not detected in the anaerobic
portion of
a
semi-solid thioglycollate medium. Growth
is good at NaCl concentrations up to 7.5
YO.
Optimum
growth temperature is 35 "C. Culture growth does not
cause haemolysis of horse, bovine or sheep blood. The
cell wall does not contain teichoic acid. Acetoin is not
produced and nitrates are not reduced. DNase, pyrroli-
donylarylamidase and #?-glucosidase activities are
negative, except that strain DD 9350T is weakly
positive for DNase and /3-glucosidase activities. Staph
Latex agglutination is negative, except that strain DD
11654 is weakly positive for this test. All strains are
resistant to novobiocin and susceptible to lysostaphin.
Acid is produced aerobically from glycerol and
#?-D-
fructose. All strains, except DD 9347, produce acid
from D-mannitol. Acid is not produced from a-lactose
and, with the exception of strain DD 9347, is not
produced from D-ribose. Characteristics of
M.
equipercicus
that are general properties of the genus
are listed above. The variable characteristics for this
species are listed in Table 4. The major API STAPH-
IDENT profile is 2000 (68
YO).
There are ten different
ID32
STAPH profiles; the most common ones begin
with the digits 17 (27
YO)
or 35 (1 8
YO)
in the nine-digit
profile, although no specific profile is represented by
more than 14% of the strains. The major ribotype
pattern types are dd 11639 (41
YO)
and dd 09345
(23
YO).
The
G
+
C content of the DNA is 45 mol
YO.
This species has a preference for perissodactyls (e.g.
horses and ponies) and is commonly found as large
populations on the skin of these mammals.
Description
of
the type strain.Type strain is ATCC
51831T
(=
DD 9350T), isolated from the skin of an
Irish thoroughbred horse. It has all of the general
properties of the species and genus described above
and also the following characteristics. Cells are spheri-
cal, 1.3-2.3 pm in diameter, have an irregular surface
(SEM) exhibiting numerous small piliform pro-
jections, and occur predominantly in tetrads. Colonies
on P agar and TSA are circular,
8
mm in diameter,
entire, convex, and with a dull matt surface and
butyrous consistency. Light- to medium-orange pig-
mentation. Marginally facultatively anaerobic; no
visible growth in anaerobic portion of thioglycollate
medium. No haemolysis of sheep, horse and bovine
blood. Positive aesculin hydrolysis and urease activity.
Weakly positive DNase and /?-glucosidase activities.
Negative latex agglutination. Acid produced aerobic-
ally from D-trehalose, D-mannitol, glycerol and
#?-D-
fructose. Acid produced weakly from maltose.
No
acid
produced from sucrose, a-lactose and D-ribose.
Sus-
ceptible to oxacillin. Type dd 09345 ribotype pattern.
lnterna tional Journal
of
Systematic Bacteriology
48
873
W.
E.
Kloos and others
Cell wall peptidoglycan is ~-Lys-Gly,-,, L-Ser. No
detectable teichoic acid. Lipoteichoic acid is present.
G
+
C content of DNA is 45 mol
YO.
Description
of
Macrococcus
bovicus
sp. nov.
Macrococcus
bovicus
(bov.ic'us. Gr. n.
bou
cow. L.
gen. n.
bovis
of a cow. M.L. adj.
bovicus
pertaining to
a bovine or cow, from which this organism was first
isolated).
The description below is based on the characteristics of
ten strains (Table 1) isolated in 1992-1994 from the
skin of a cow, a pony and several horses. Colonies
grow to 4+ 1 mm in diameter on P agar and TSA.
They are slightly convex, entire, butyrous, glistening
and opaque, and have a pale-yellow to medium-orange
pigmentation. Growth is not detected in the anaerobic
portion of a semi-solid thioglycollate medium. Growth
is good at NaCl concentrations up to 7.5
YO.
Optimum
growth temperature is 35 "C. Culture growth causes a
partial haemolysis (greening) of horse and bovine
blood. The cell wall does not contain teichoic acid.
Lipoteichoic acid is present. Acetoin is not produced.
Pyrrolidonylarylamidase activity is negative. All
strains are resistant to novobiocin and susceptible to
lysostaphin and oxacillin. Acid is produced aerobically
from D-mannitol, glycerol and P-D-fructose. Acid is
not produced from a-lactose. Characteristics of
M.
bovicus
that are general properties of the genus are
listed above. The variable characteristics of this species
are listed in Table 4. Major API STAPH-IDENT
profiles are 0200 (30
%),
4500 (20
YO)
and
4600
(20
%),
and major
ID32
STAPH profiles are 062200000
(20
%),
0623 10200 (20
%)
and 072300200 (20
YO).
The
major ribotype pattern type is dd 09344 (40%). The
G+C content of the DNA is 42-44 mol%. This
species appears to have a preference for perissodactyls
(e.g. horses and ponies) and artiodactyls (e.g. cattle).
Description
of
the type strain.
Type strain is ATCC
51825T
(=
DD 4516T), isolated from the skin of a
Holstein cow.
It
has all of the general properties of the
species and genus described above and also the
following characteristics. Cells are spherical, 1.2-
2.1 pm in diameter, have a slightly irregular surface
(SEM), and occur singly and in pairs, tetrads and short
chains. Colonies on P agar and TSA are circular,
3-5 mm in diameter, entire, slightly convex, and with a
glistening surface and butyrous consistency. Medium-
orange pigmentation. Marginally facultatively anaer-
obic; no visible growth in anaerobic portion of
thioglycollate medium. Partial haemolysis (greening)
of horse and bovine blood. Positive DNase activity
and latex agglutination. Negative aesculin hydrolysis,
nitrate reduction and urease activity. Acid produced
aerobically from D-trehalose, D-mannitol, glycerol and
p-D-fructose. No acid produced from sucrose, a-
lactose, maltose and D-ribose. Type dd 04516 ribotype
pattern. Cell wall peptidoglycan is ~-Lys-Gly,, L-Ser.
No detectable teichoic acid. Lipoteichoic acid is
present.
G
+
C content of DNA is 44 mol
%.
Description
of
Macrococcus
carouselicus
sp. nov.
Macrococcus
carouselicus
(car.ou.se1'i.cus. M.L. adj.
carouselicus
pertaining to a carousel or merry-go-
round, which has carousel horses).
The description below is based on the characteristics of
18 strains (Table 1) isolated in 1993-1995 from the skin
of horses and ponies. Colonies grow to
5
+
2 mm in
diameter on P agar and 7& 1 mm on TSA. They are
slightly convex, entire, butyrous, glistening and
opaque, and have a cream- to light-orange pigmen-
tation. Growth is not detected in the anaerobic portion
of a semi-solid thioglycollate medium. Growth is good
at NaCl concentrations up to 7.5
%.
Optimum growth
temperature is 35 "C. Culture growth usually does not
cause haemolysis of horse and sheep blood. The cell
wall does not contain teichoic acid. Lipoteichoic acid is
present. Acetoin is not produced and, with the ex-
ception of strain DD 9346, nitrates are not reduced.
All strains, except DD 934
1,
hydrolyse aesculin.
DNase activity is positive. All strains, except DD 9607,
are negative for P-glucosidase activity. Pyrrolidonyl-
arylamidase activity is negative. All strains are re-
sistant to novobiocin and susceptible to lysostaphin
and oxacillin. All strains, except DD 9341, weakly
produce acid aerobically from P-D-fructose. Acid is not
produced from maltose, D-ribose and a-lactose.
Characteristics of
M.
carouselicus
that are general
properties of the genus are listed above. The variable
characteristics of this species are listed in Table 4. The
major API STAPH-IDENT profile is 0000 (94
YO)
and
major ID32 STAPH profiles are 050100000 (28%),
070100000 (28%) and 010100000 (22%). The major
ribotype pattern types are dd 09342 (28%) and dd
09349 (17%). The G+C content of the DNA is
41 mol
%.
This species has a preference for perisso-
dactyls (e.g. horses and ponies) and is commonly
found as large populations on the skin of these
mammals.
Description
of
the type strain.
Type strain is ATCC
51828T
(=
DD 934ST), isolated from the skin of an
Irish thoroughbred horse. It has all the general
properties of the species and genus described above
and also the following characteristics. Cells are spheri-
cal, 14-25 pm in diameter, have a slightly irregular
surface (SEM), and occur in pairs, tetrads, short chains
and clusters. Colonies are
4-5
mm in diameter on
P
agar and 6-7 mm in diameter on TSA, circular, entire,
slightly convex, and with a glistening surface and
butyrous consistency. Cream-orange pigmentation.
Marginally facultatively anaerobic
;
no visible growth
in anaerobic portion of thioglycollate medium. No
haemolysis of sheep, horse and bovine blood. Positive
aesculin hydrolysis and latex agglutination. Negative
nitrate reduction and P-glucosidase activity. Acid
produced aerobically from sucrose. Acid produced
weakly from D-trehalose, glycerol and P-D-fructose.
No acid produced from D-mannitol, maltose, D-ribose
and a-lactose. Type dd 09342 ribotype pattern. Cell
wall peptidoglycan is ~-Lys-Gly,_,, L-Ser. No detect-
874
In terna tiona I
lo
urna
I
of
Systematic Bacteriology
48
Macrococcus
gen. nov.
Table
5.
Characteristics useful
for
differentiating
Macrococcus
species
from
the related genus
Staphylococcus
and
from
each other
The phylogenetic relationship of the genus
Macrococcus
to the genus
Staphylococcus
was estimated on the basis
of
16s rDNA
sequences.
+
,
90
%
or more
of
the strains are positive;
,
90
%
or more
of
the strains are weakly positive; d,
1
1-89
%
of the
strains are positive;
-,
90
YO
or more
of
the strains are negative. Parentheses indicate a delayed response.
Characteristic
M.
caseolyticus
M.
equipercicus
M.
bovicus
M.
carouselicus Staphylococcus
For
differentiating
Cell diameter 2 2 mm
Cell wall teichoic acid
Oxidase
DNA G+C
>
40
mol%
NotI
digestion of DNA$
Genome size
2
2000 kb
Dull orange
For
differentiating
Haemol ysis
Acetoin production
Aesculin hydrolysis
Nitrate reduction
DNase
Urease
Pyrrolidonylar ylamidase
Staph Latex
Acid (aerobic) produced
genera
(Microdase disk)
pigmen tation
species
(horse blood)
agglutination
from
:
D-Mannitol
Maltose
Sucrose
Glycerol
D-Ribose
a-Lactose
P-D-Fructose
+
+
-
+