TAXONOMY Back to top
Gram-positive anaerobic cocci comprise a diverse
group of organisms. Until recently, most
clinical isolates of gram-positive anaerobic cocci
were identified as species in the
genus Peptostreptococcus. Peptostreptococcuswas
described as a genus in 1936 and was
considered the anaerobic equivalent of Streptococcus.
It comprised 16 recognized species,
with a G+C range from 27 to 37 mol%, except for Peptostreptococcus
productus (44 to 45
mol%). In the past decade, molecular techniques
such as DNA-DNA hybridization and 16S
rRNA gene sequencing have been widely employed in
elucidating evolutionary relationships
among gram-positive anaerobic coccus species both
within and between genera. Most
recently, genome sequences of Finegoldia magna strain
ATCC 29328, Anaerococcus
prevotii strain DSM 20548, and Atopobium parvulum strain DSM 20469
have been published
(http://www.ebi.ac.uk/2can/genomes/bacteria.htm).
Polyphasic taxonomic studies have indicated that
the gram-positive anaerobic cocci vary
markedly in fundamental characteristics, and a
number of new genera,
including Anaerococcus, Finegoldia, Gallicola,
Parvimonas,
Peptoniphilus, and Murdochiella, have been proposed (39,
84, 125, 128). Now the
genusPeptostreptococcus contains only two
species, the type species, P. anaerobius, and a
newly described species,Peptostreptococcus
stomatis (33). Peptostreptococcus
magnus and Peptostreptococcus micros were placed in two new
genera, Finegoldia and Micromonas,
respectively (83). However, the name “Micromonas” is
illegitimate because of precedence of a
microalga Micromonas; subsequently, Parvimonas
micra was proposed as a replacement
for “Micromonas micros” (125).
Ezaki et al. (39) proposed three other
genera: Anaerococcus,which includes the
saccharolytic, butyrate-producing species (A.
hydrogenalis, A. lactolyticus, A. octavius, A.
prevotii, A. tetradius, A.
vaginalis); Peptoniphilus, which contains the nonsaccharolytic,
butyrate-producing
species (P. asaccharolyticus, P. harei, P.
lacrimalis, P. indolicus, and P.
ivorii), and Gallicola, which contains a single species, G. barnesae.
Song et al. (119)
described two novel species of Peptoniphilus,
P. gorbachii and P. olsenii, and one
of Anaerococcus, A. murdochii, isolated
from clinical specimens. Most
recently, Murdochiella, a novel genus which
includes a single species (Murdochiella
asaccharolytica) was proposed. Peptococcus is remotely
related to other species of grampositive
anaerobic cocci and is rarely cultured from human
clinical specimens. Peptococcus
niger is now the sole remaining representative of this genus.
The taxonomy of other validly published
gram-positive anaerobic cocci from human clinical
specimens, such asStreptococcus parvulus,
Peptostreptococcus
productus, and Peptostreptococcus saccharolyticus, has also undergone
revision.
Streptococcus parvulus has been transferred to the genus Atopobium as
Atopobium
parvulum (27). P. productus was reclassified as Ruminococcus
productus by Ezaki et al. (38),
and a novel genus was recently proposed for this
organism, Blautia (Blautia
producta) (68); Peptostreptococcus saccharolyticus has
been transferred to the
genus Staphylococcus based on an analysis
of nucleic acid relatedness data and cell wall peptidoglycan
structure. Table 1 shows the changes in classification of gram-positive
anaerobic coccal species
from human clinical specimens.
The
gram-negative anaerobic cocci are currently classified in five genera of the
family
Veillonellaceae, the genera Veillonella,
Acidaminococcus,
Megasphaera, Anaeroglobus, and Negativicoccus (24, 57, 72).
The
familyVeillonellaceae,
formerly “Acidaminococcaceae,” is currently classified in the
phylum
Firmicutes (low-G+C-content gram-negative bacteria) and the class Clostridia
(72).
The
family Veillonellaceae has been removed from the classification in the
latest edition
of Bergey’s
Manual of Systematic Bacteriology (45, 72).
The
genus Veillonella is widely distributed in the oral, genitourinary,
respiratory, and
intestinal
biotas of humans and animals. It is subdivided into 10 species: Veillonella
atypica,
Veillonella
caviae, Veillonella criceti, Veillonella denticariosi, Veillonella dispar,
Veillonella
montpellierensis,
Veillonella parvula, Veillonella ratti, Veillonella rodentium, and Veillonella
rogosae
(56). Of these, only V.atypica, V.denticariosi, V.dispar,
V.parvula,
and V.rogosaehave been isolated from human oral cavities (2, 4, 23, 62).
Four
other
species, V. caviae, V. criceti, V. ratti, andV. rodentium, were
found only in animals,
except
for one V. ratti isolate, which was recovered from human semen.
Besides
Veillonella spp., A. fermentans and A. intestini of the
genus Acidaminococcus, M.
elsdenii
and M. micronuciformis of the genus Megasphaera, and
A. geminatus of the
genus
Anaeroglobus have been isolated from human clinical samples.
DESCRIPTION OF THE GROUP Back
to top
The
organisms included in this chapter are obligately anaerobic, non-spore-forming,
sometimes
elongated cocci. The genera Anaerococcus, Anaerosphaera, Finegoldia,
Gallicola,
Parvimonas,
Peptococcus, Peptoniphilus, andPeptostreptococcus, as
well as the newly
described
taxon Murdochiella (128), are gram-positive,
coccobacillary, or, occasionally,
coccoid
cells. In Gram-stained preparations of pure cultures, cells vary in size from
0.3 mm
to
2.0 mm and can be arranged in pairs, short chains, tetrads, small clusters, or
irregular
masses;
most species are present either as chains or as clumps. The ability to utilize
carbohydrates
varies greatly; some genera are asaccharolytic, but a few are strongly
saccharolytic.
For most species, the products of protein digestion appear to be the principal
energy
source. The genus Staphylococcus contains two species, S.
saccharolyticus and S.
aureus
subsp. anaerobius, which initially grow under anaerobic
conditions and become
aerotolerant
on subcultures (see chapter 19 in this Manual). Strictly
anaerobic
Staphylococcus epidermidis is reported to be occasionally isolated from
clinical
specimens
(105). The genera Veillonella, Acidaminococcus,
Megasphaera,Anaeroglobus,
and Negativicoccus (72)
are gram-negative cocci. Cells vary in
size
from 0.3 μm to 2.5 μm. They characteristically occur in pairs, but single
cells, masses,
or
chains may also occur. Carbohydrates are weakly fermented or not fermented. Gas
is
produced.
The metabolic end products are the principal characteristics by which the
genera
can
be differentiated.
EPIDEMIOLOGY AND TRANSMISSION Back
to top
Gram-positive
anaerobic cocci are part of the normal biota of the mouth, upper respiratory
and
gastrointestinal tracts, female genitourinary system, and skin (82).
Gram-positive
anaerobic
cocci constitute 1 to 15% of the normal oral biota (124); Parvimonas
micra is
usually
considered to be the predominant species of gram-positive anaerobic cocci in
the oral
biota,
and P. anaerobius and F. magna have been reported to be present. The
gastrointestinal
tract hosts a wide variety of gram-positive anaerobic cocci, including most
recognized
species of Peptostreptococcus. B. producta is one of the most
common organisms
in
the gastrointestinal biota; F. magna and A. prevotii are also
common. Murdochiella is also
presumably
found in the bowel (128). Several other gram-positive anaerobic cocci are found
less
often. Large numbers of gram-positive anaerobic cocci can be found in the
female
genitourinary
tract. A. tetradius, A. lactolyticus, and A. vaginalis were first
described from
vaginal
discharges (36, 37, 67); P. anaerobius, P. asaccharolyticus, P. hydrogenalis, F.
magna,
Parvimonas micra, A. prevotii, and Peptococcus niger have
also been isolated from
that
site. The skin biota contains gram-positive anaerobic cocci; F. magna is
the species
identified
most frequently, followed by P. asaccharolyticus (82).
Gram-negative
anaerobic cocci form part of the oral, genitourinary, respiratory, and
intestinal
biota of humans.Veillonella species are part of the normal mouth, upper
respiratory
tract,
gastrointestinal tract, and vaginal biotas; Acidaminococcus and Megasphaera
are part
of
the intestinal biota. A. geminatus has also been isolated from the human
mouth and
gastrointestinal
tract (55).
CLINICAL SIGNIFICANCE Back
to top
Estimation
of the clinical significance of anaerobic cocci isolated from clinical
specimens is
often
difficult partly due to their recovery from poorly obtained specimens (failure
to exclude
normal
biota). Anaerobic gram-positive cocci are opportunistic pathogens and comprise
approximately
one-quarter of all isolates from anaerobic infections (81, 82).
They may be
present
in a great variety of infections involving all areas of the human body, ranging
in
severity
from mild skin abscesses to more serious and life-threatening infections, such
as
brain
abscess, bacteremia, necrotizing pneumonia, and septic abortion. Brain abscess
and
meningitis
are among the more serious infections involving anaerobic cocci (1, 64, 70).
In
deep-space
head and neck infections (13), 9.2% of anaerobic
isolates were gram-positive
anaerobic
cocci (F. magna, P. micra, and P. anaerobius). The incidence of
anaerobic cocci in
pleuropulmonary
infections, such as lung abscess, necrotizing pneumonia, aspiration
pneumonia,
and empyema, is about 40% (73, 129).
Anaerobic cocci are often isolated with
other
organisms in skin and soft tissue infections, including progressive bacterial
synergistic
gangrene,
necrotizing fasciitis, diabetic foot ulcer, and crepitant cellulitis (10, 82, 86, 123).
Other
infections in which anaerobic cocci have been recognized as significant
pathogens are
infections
of the female genital tract and intra-abdominal infections (21, 31, 40, 116).
In a
recent
study showing a decrease in anaerobic bacteremia, gram-positive anaerobic cocci
increased
from 5.4% in an early period to 12% (40).
Although most infections involving
gram-positive
anaerobic cocci are polymicrobial (41),
there are many instances of their
isolation
in pure culture (81, 82); most relate to F. magna, but there are also reports of P.
anaerobius,
P. asaccharolyticus, P. indolicus, P. micra, A. vaginalis, and P.
harei in pure
culture.
F.
magna is the most pathogenic and one of the most frequently isolated
gram-positive
anaerobic
coccal species found in human clinical specimens. Possible F. magna pathogenicity
factors
have been identified, including capsule formation (16)
and production of various
enzymes,
such as collagenase, gelatinase (65),
and subtilisin-like serine-proteinase (SufA)
(59, 60). F.
magna was also found to express surface proteins, such as protein L, which
is a
B-cell
superantigen (8), the albumin-binding protein PAB (30),
and a protein designated FAF
(F.
magna adhesion factor) (44); these proteins may play
an important role in creating an
ecologic
niche for F. magna, decreasing antibacterial activity, and suppressing
angiogenesis
and
thus providing an advantage for the survival for this opportunistic pathogen. F.
magna
has been isolated from a wide variety of infections at various
body sites in pure
culture.
These include cases of endocarditis (43)
and meningitis and pneumonia (82, 89),
some
of which have been fatal. F. magna is most commonly associated with
infections of
skin
and soft tissue, bones, and joints but has also been isolated from cases of
septic
arthritis
(42), prosthetic implant infections (29),
breast abscess (34), diabetic foot infections
(54),
bacterial vaginosis, and upper respiratory tract infections, such as sinusitis
and otitis
media.
P.
anaerobius is involved in polymicrobial infections, including abscess of the
brain, ear, jaw,
pleural
cavity, pelvis, urogenital tract, external genitalia, abdominal regions, and
nasal
septum
(17, 18, 52). The isolation ofP. anaerobius from endocarditis
specimens has been
reported
(77). P. anaerobius has been associated with gingivitis (78)
and periodontitis (130);
it
is one of the species found most frequently in the root canals of teeth with
periapical
abscess
and has been isolated from a peritonsillar abscess (26, 103).
P.
micra is increasingly recognized as an important oral pathogen (88).
It has been shown to
produce
collagenase, hemolysin, and, occasionally, elastase, all virulence factors (88).
Although
it is considered a natural commensal of the oral cavity (85, 91),
elevated counts of
this
organism are associated with periodontal destruction (96).
It is also commonly isolated
from
other oral infections, such as endodontic lesions and peritonsillar infections
(76). P.
micra is not restricted to the oral cavity; it is often isolated in mixed
anaerobic
infections
from different body sites, including brain abscess, otitis media, sinus
infection,
human
bite wounds, pleural empyema, intra-abdominal infection, anorectal abscess,
septicemia,
gynecological infection, vertebral osteomyelitis, and prosthetic joint
infection
(82).
Anaerobic
gram-negative cocci account for a very small percentage of the anaerobic cocci
isolated
from human specimens (6). Veillonella sp. strains are frequently isolated from
clinical
specimens in aerobic-anaerobic polymicrobial cultures. Rarely, Veillonella species
have
been the only etiologic agents identified in serious infections such as
meningitis,
osteomyelitis,
prosthetic joint infection, pleuropulmonary infection, endocarditis, and
bacteremia
(5, 19, 20, 69, 71, 73, 113). In most clinical reports of Veillonella infection, the
isolates
have not been identified to the species level. There have been only four
previous
reports
of confirmed V. parvuladiscitis or vertebral osteomyelitis (75, 113).
Although
the spectrum of infections has remained relatively unchanged since the
extensive
review
by Murdoch in 1998 (82), the prevalence of these organisms as pathogens is clearly
increasing.
COLLECTION, TRANSPORTATION, AND STORAGE OF
SPECIMENS Back to top
Most
gram-positive anaerobic cocci isolated from human clinical material are not
extremely
oxygen
sensitive. Specimens suspected of harboring anaerobic cocci should be
collected,
transported,
and stored by methods outlined elsewhere (see chapter 19 in
this Manual).
DIRECT EXAMINATION Back
to top
Microscopy
In
clinical samples, direct Gram staining shows that anaerobic cocci vary in size
and occur in
chains,
in pairs, or singly. P. micra cells are less than 0.6 μm in diameter and
occur in
packets
and short chains; other anaerobic cocci, such as A. tetradius, A. prevotii, and
F.
magnus,
have cells greater than 0.6 μm in diameter in pairs and clusters
and may resemble
staphylococcal
cells. The difference in cell size has been used as one characteristic to
distinguish
between P. micra and F. magna.
Antigen Detection
Serological
studies described an indirect fluorescent- antibody test for P. anaerobius,
P.
micra,
and B. producta(28),
but they were not taken further.
Nucleic Acid Detection
Molecular
diagnostics of infectious diseases, in particular nucleic-acid-based methods,
is the
fastest-growing
field in clinical laboratory diagnostics. These applications are beginning to
replace
or complement culture-based, biochemical, and immunological assays in
microbiology
laboratories. Molecular methods, such as nucleic acid probe hybridization and
PCR
amplification, are not yet standardized or available commercially for the
direct
demonstration
of medically important gram-positive anaerobic cocci from clinical specimens.
However,
several studies have used molecular techniques to identify and detect anaerobic
cocci.
DNA probes targeting the 16S rRNA gene have been used to detect P.
anaerobius
and P. micra, and PCR assays specific for detection of F.
magna, P.
anaerobius,
and P. micra directly from clinical specimens have been
developed
(98, 99, 100, 101,114, 117).
More recently, a real-time PCR technique has been applied for
quantitative
detection of anaerobic cocci. It has been used to detect P. micra from
prosthetic
joint
infection, endodontic infections, and periradicular lesions (3, 9, 11, 12, 49, 87, 122).
Marrazzo
et al. (74) reported using real-time PCR to detect P.
lacrimalis
and Megasphaera spp. in vaginal samples. The same approach
has also been
applied
to detectVeillonella spp. in human oral and lower intestinal samples, as
well as
samples
from asymptomatic vaginal infections and endodontic infection (7, 95, 102).
Several
studies
reported using a checkerboard DNA-DNA hybridization method to directly detect
microbes
from oral clinical samples, including P. micra (92, 93, 106, 109,115).
ISOLATION PROCEDURES Back
to top
Routinely
used anaerobic plate media, such as brucella, Columbia, or Schaedler agar base
supplemented
with 5% sheep blood, vitamin K1, and hemin, support the growth of these
microorganisms.
However, CDC (Centers for Disease Control and Prevention) agar base
(see
chapter 17 in this Manual) gives better recovery of gram-positive
anaerobic cocci than
brucella
agar or other agars. The usual procedures for anaerobes should be followed (55).
Many
of these organisms require a high moisture content for optimal growth, so fresh
media
should
be used. Laboratories unable to prepare their own media may wish to consider
the
use
of commercially prepared, prereduced, anaerobically sterilized (PRAS) blood
agar
(Anaerobe
Systems, Morgan Hill, CA). These media have an extended shelf life of up to 6
months
and yield results comparable to or better than those obtained with fresh media.
Gram-positive
anaerobic cocci are heterogeneous; a single medium is unlikely to support the
growth
of all representatives and be reasonably selective. Wren (134)
showed that nalidixic
acid-Tween
80 blood agar gave better isolation than neomycin blood agar, possibly due to
the
particularly inhibitory nature of neomycin against gram-positive anaerobic
cocci, but
recommended
that a combination of different media be used to maximize recovery rates.
Petts
et al. (94) reported that oxolinic acid was superior to nalidixic acid for
suppression of
staphylococci,
while permitting the growth of nonsporing anaerobes, including gram-positive
anaerobic
cocci. Turng et al. (126) described a selective and differential medium
for Parvimonas
micra that contains colistin-nalidixic acid agar (Difco, Detroit, MI),
which is a
selective
base for gram-positive cocci supplemented with glutathione and lead acetate.
Strains
of Parvimonas micra can use the reduced form of glutathione to form
hydrogen
sulfide,
which reacts with lead acetate to form a black precipitate under the colony.
Tween
80
supplementation (0.5%) of media may improve the growth of some gram-positive
anaerobic
cocci.
A
recent study (50) tested different media for recovery of Veillonella spp. from
saliva
samples
and concluded that a selective medium for Veillonella with vancomycin
and laked
blood
gave the greatest recovery ofVeillonella. This medium can also be used
for
presumptive
identification of Veillonella, since the colonies produce a red
fluorescence at a
wavelength
of 365 nm.
IDENTIFICATION Back to top
Phenotypic Tests
Some
gram-positive anaerobic cocci, particularly strains of P. asaccharolyticus, decolorize
readily
with Gram stain and can be confused with gram-negative anaerobes, such as
veillonellae.
Gram-positive anaerobic cocci can be distinguished from gram-negative
anaerobic
cocci by special-potency disks (vancomycin, 5 μg; kanamycin, 1,000 μg; and
colistin,
10 μg) (55). Generally, gram-positive anaerobic cocci are sensitive to vancomycin
and
resistant to colistin, whereas the gram-negative anaerobic cocci are resistant
to
vancomycin.
The cell morphology of older cultures of gram-positive anaerobic cocci can be
very
irregular, with many coccobacillary and rod-like forms. It is also important to
distinguish
gram-positive
anaerobic cocci from microaerophilic organisms, such as strains
of Streptococcus
species. A simple and reliable test is to apply a 5-μg metronidazole disk
to
the
edge of the inoculum; gram-positive anaerobic cocci show a zone of inhibition
of 15 mm
or
larger, whereas microaerophilic strains show no zones after incubation for 48 h
(80).
P.
anaerobius is the only gram-positive anaerobic coccus that gives a zone of
inhibition of
≥12
mm around a sodium polyanethol sulfonate (SPS) disk. Parvimonas micra also
exhibits a
zone
of inhibition with SPS; however, the zone is usually <12 mm in size. Most P.
anaerobius
strains form distinctive colonies on enriched blood agar; they are
1 mm in
diameter
after 24 h, they are gray with slightly raised off-white centers, and they have
a
distinctive
sweet odor (80). Parvimonas micra and F. magna can be readily
distinguished by
a
combination of colonial morphology and proteolytic enzyme profiling, supported
by Gramstained
cell
morphology to assess the cell size. An anaerobic coccus with a milky halo
around
the
colonies on blood agar and small cells (<0.6 μm) can be presumptively
identified
as Parvimonas
micra (80, 129). F. magna cells are larger than those of most
peptostreptococci.
Published data (83) also indicate that they can be differentiated by
enzymatic
tests for proteolytic activity, such as proline arylamidase, phenylalanine
arylamidase, and tyrosine
arylamidase (see Fig. 1).
A.
prevotii and A. tetradius were reported as common species of
gram-positive anaerobic
cocci
in human clinical material in early surveys. However, nucleic acid studies
indicate that
they
are very heterogeneous. Again, our study based on 16S rRNA gene sequencing
indicated
that a large percentage of organisms identified as A. prevotii/A.
tetradius are
strains
of A. vaginalis. It is likely that strictly defined strains of A.
prevotii/A. tetradiusare
only
occasionally recovered from most clinical specimens. The activity of
pyroglutamic acid
arylamidase
might be useful for differentiation of A. prevotii and A. tetradius;
however,
distinctions
cannot be generalized because insufficient numbers of strains of each species
have
been reliably identified. A. prevotii and A. tetradius can be
distinguished from other
recognized
species of gram-positive anaerobic cocci by production of α-glucosidase and β-
glucuronidase.
Strains of other saccharolytic gram-positive anaerobic cocci such as A.
vaginalis
and A. lactolyticus can be differentiated by their enzyme
profiles (118).
Table
2 summarizes the differential characteristics of gram-positive
anaerobic coccal species.
Based
on published data from our group and others (118),
we developed a flow chart for
rapid
identification of gram-positive anaerobic cocci (Fig. 1).
The identification is based on
phenotypic
tests that can be performed in any diagnostic laboratory. Most of the
information
presented here relates to
the phenotypic characteristics of strains isolated from humans.
Several
identification systems, such as the Rapid ID 32A (bioMerieux, Marcy L’Etoile,
France)
and
RapID ANA II (Remel, Inc., Lenexa, KS) systems, are available commercially for
the
rapid
identification of anaerobes. Evaluations of these biochemical kits indicate
that they may
be
of value for characterizing anaerobic cocci; however, these systems are
designed to
identify
as wide a range of anaerobes as possible, and they contain many tests of little
relevance
for identification of anaerobic cocci. Furthermore, databases accompanying the
kits
are
often incomplete or inaccurate, especially with a number of newly described
species. Our
most
recent evaluation of the Rapid ID 32A kit for identification of gram-positive
anaerobic
cocci
by comparison with 16S rRNA gene sequencing identification showed that the
system is
good
for accurate identification of Parvimonas micra, P. anaerobius, F. magna, and
P.
asaccharolyticus
but not other species (unpublished data).
Gram-positive
anaerobic cocci are separated into five groups based on fatty acid end
products
of metabolism as analyzed by gas-liquid chromatography (GLC) (Table
2): (i) an
acetate
group (containing F. magna andParvimonas micra) that produces
only acetic acid, (ii)
a
butyrate-acetate group (containing all of the species in the genus Anaerococcus)
that
produces
butyric acid as its major terminal volatile fatty acid (VFA) and acetic acid as
a
second
major acid, (iii) an acetate-butyrate group (containing all of the species in
the
genusPeptoniphilus
except P. ivorii) that produces acetic acid as its major terminal
VFA and
butyric
acid as the second acid, (iv) a caproate group whose members produce large
quantities
of longer-chain VFAs, and (v) an isovaleric acid group (containing P. ivorii,
the
only
species of gram-positive anaerobic cocci that produces a major terminal peak of
isovaleric
acid). The most important species in group iv is P. anaerobius, the only
species of
gram-positive
anaerobic cocci to produce a major terminal peak of isocaproic acid. GLC is
also
useful for identifying the rarely isolated Peptococcus niger and P.
octavius, which
produce
n-caproic acid.
Veillonella,
Acidaminococcus, and Megasphaera comprise the principal genera of anaerobic
gram-negative
cocci. The identification of Veillonella at the species level remains
uncertain
and
inconvenient owing to the lack of conventional phenotypic and biochemical
discriminating
tests (62). Moreover, serological groupings (104)
are no longer
available.
Table 3 contains a key for differentiating the genera of anaerobic
gram-negative
cocci.
Molecular Methods
Direct sequencing of 16S rRNA genes (rrs) has
proven to be a stable and specific marker for
bacterial identification. Two groups have
evaluated the utility of 16S rRNA gene sequencing
as a means of identifying clinically important gram-positive
anaerobic cocci (118, 119, 134).
Our studies (118, 119)
indicated that problems exist in the public database; for example,
among the 13 type strains of gram-positive
anaerobic coccal species that we tested, only
four “perfectly” matched their corresponding
sequences in GenBank, whereas the other nine
had lower sequence similarities (<98%). This is
due mainly to the poor quality of bacterial
sequences deposited in GenBank from early years.
Based on the correct 16S rRNA sequences
that we deposited in GenBank, a multiplex-PCR
scheme was developed for rapid
identification of clinically significant
gram-positive anaerobic coccal species (120). More
recently, Wildeboer-Veloo et al. (132)
developed 16S rRNA-based probes for the
identification of gram-positive anaerobic cocci
isolated from human clinical specimens.
However, these assays have not been evaluated for
direct bacterial detection from clinical
samples.
The MicroSeq 16S rRNA sequencing system could
identify only 39% of 23 anaerobic cocci
(133). A commercial real-time PCR setup (Septi-Fast;
Roche), which is multiplexed for 25
pathogens, does not cover anaerobic cocci at this
time (66). There have been favorable
reports regarding the Vitek 2 system with an
anaerobe identification card but with limited
numbers of species and strains of anaerobic cocci
(97). Matrix-assisted laser desorption-time
of flight mass spectrometry is a promising tool,
but the database is limited for gram-positive
anaerobic cocci; published studies have involved
only a few strains of anaerobic grampositive
cocci and did not detect them well (112).
Beighton et al. (4) reported using rpoB gene
sequencing for identifying Veillonella spp.
isolated from tongue samples because 16S rRNA
sequence analysis does not reliably
differentiate among all members of this genus.
Subsequently, a simple two-step PCR
procedure was developed for the identification of
the recognized oralVeillonella species (53).
TYPING SYSTEMS Back to top
Molecularly based methods such as PCR
amplification of 16S rRNA genes followed by
restriction fragment length polymorphism analysis
has proved to be useful for gram-positive
anaerobic cocci (100) and Veillonellaspecies
strain typing (110, 111). PCR-amplified rRNA
gene spacer polymorphism analysis was also successfully
applied in the rapid differentiation
of the currently recognized taxa within the group
of anaerobic gram-positive cocci (51).
ANTIMICROBIAL SUSCEPTIBILITIES Back to top
New information regarding the antimicrobial
susceptibilities of gram-positive anaerobic cocci
(Table 4) is sparse compared with the information
available for other anaerobic species.
Penicillins
are considered to be an effective first-line therapy for gram-positive
anaerobic
cocci.
Most evidence suggests that P. asaccharolyticus, F. magna, and P.
micra are usually
susceptible
to penicillins, although Wren (134) reported 16% and 8%
resistance to penicillin
among
isolates of F. magna and Parvimonas micra,respectively, in their
study.
Cephalosporins
are usually, but not always, effective. Carbapenems are extremely active.
Several
authorities maintain that almost all gram-positive anaerobic cocci are
susceptible to
metronidazole,
but resistance has frequently been reported. Strains that are microaerophilic
(and
therefore streptococci) are much more likely to be resistant to metronidazole.
Susceptibility
to clindamycin varies widely; local geographic variation should be taken into
account.
A French multicenter study (79) reported 28% clindamycin
resistance
among
Peptostreptococcus spp., and an American study by Sanchez et al. (108)
noted >10%
resistance
of F. magna to clindamycin. Wren (134)
reported 9% of F. magna strains to be
resistant
to clindamycin in London, United Kingdom. Erythromycin and two other
macrolides,
clarithromycin
and azithromycin, have similar efficacies and are probably not active enough
to
be recommended. Several studies (90, 134)
indicated that older quinolones, such as
ciprofloxacin,
have only moderate activity, but more recently developed agents are
extremely
active (46, 90, 131). In another recent study, Koeth et al. (61)
reported that the
rates
of susceptibility of F. magna to levofloxacin and clindamycin were 72.4%
and 84.7%,
respectively.
It is highly desirable that future investigations present data on different
species
separately.
A recent study by Brazier et al. (15)
presented data on different gram-positive
anaerobic
coccal species separately. They found that the highest percentage of overall
resistance
detected among gram-positive anaerobic cocci was 41.6% resistance to
tetracycline,
followed by 27.4% resistance to erythromycin. Among gram-positive anaerobic
cocci
as a group, 7.1% of isolates were resistant to penicillin and clindamycin and
3.5% of
isolates
were resistant to amoxicillin-clavulanate. There was no resistance among
grampositive
anaerobic
cocci to piperacillin-tazobactam, chloramphenicol, cefoxitin, imipenem, or
metronidazole.
A more recent study by the same group found an overall resistance rate of
7%
of gram-positive anaerobic cocci to penicillin and/or clindamycin (14).
Similar results
have
been reported previously (15), although a higher
prevalence of resistance, in particular
to
clindamycin, has been reported in some studies (61, 79).
In another study, one strain
of Ruminococcus
gauvreauii was found to be highly resistant to vancomycin and teicoplanin
(>256
μg/ml) (32).
It
is worth noting that the resistance rates of P. stomatis and P.
anaerobius are different
(63). P.
anaerobiussensu lato exhibits some resistance to several drugs:
amoxicillin,
amoxicillin-clavulanate
(3 of 30 strains resistant), cefoxitin (2 of 30 strains resistant), and
azithromycin
and moxifloxacin (1 of 30 strains resistant). There was no resistance found in
31
strains of P. stomatis.
Certain
newer agents would not be considered primary agents for therapy of infections
due
to
anaerobic cocci, but when they are indicated for another reason in a mixed
infection
involving
anaerobes, it is useful to know about their activity versus anaerobes. Useful
references
are available regarding the activities against anaerobes of ceftobiprole (35, 47),
oritavancin
(25), daptomycin (127), dalbavancin (48),
and tigecycline (121). A striking
example
of another use of agents such as these is the report of the successful use of
linezolid
in a patient with a brain abscess due to a Peptostreptococcus sp. after
failure of
standard
treatment (107).
EVALUATION, INTERPRETATION, AND REPORTING OF
RESULTS Back to top
Because
anaerobic bacteriology is time-consuming, several interim reports are
desirable. The
initial
report should give Gram stain results and bacterial and human cell
morphologies. The
relative
quantities of different organisms seen in the smear give a good overall
impression of
the
specimen quality, the nature of the polymicrobial infection, and the relative
importance
of
each organism. In general, bacterial isolates that are predominant and
resistant to
antimicrobial
agents should be given the greatest attention. Bacteria present in pure culture
or
in large numbers are probably of major importance, as are organisms recovered
in
multiple
cultures and isolated from normally sterile sites. Furthermore, Gram stain
results
can
guide the laboratory in choosing media for optimal recovery of the predicted
organisms.
The
significance of finding anaerobic gram-positive and gram-negative cocci in
clinical
specimens
depends on the specimen and the likelihood that it was contaminated by the
microbiota
of the skin or mucous membranes. Hence, interpretation of culture results is
dependent on the nature and
quality of the specimen submitted to the laboratory.
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