Within
the family Bacteroidaceae, the genus Bacteroides consists of
saccharolytic, bileresistant,
and
nonpigmented species, mainly isolated from the gut (178).
Currently, the
genus
is limited to species within theBacteroides fragilis group, which
includes more than 20
species
(212). Of these, especially B. fragilis, B. thetaiotaomicron, and
B. ovatus are highly
relevant
in human infections. Since 2006, few taxonomic changes have occurred within the
genus;
some novel species have been described, and some former Bacteroidesspecies
have
been
moved to other genera. Novel species of the B. fragilis group (Table
1) have been
isolated
from feces of healthy subjects (8–10, 28, 76, 96, 161, 210). B.
distasonis, B.
goldsteinii,
and B. merdae now reside in a novel genus, Parabacteroides
(167), and B.
splanchinus
is now in another novel genus, Odoribacter(74). B.
capillosus, which is both
phenotypically
and genotypically distinct from the genus Bacteroides, has been assigned
to a
novel
genus, Pseudoflavonifractor, as P. capillosus (26).
This represents a distinct lineage in
clostridial
cluster IV of the phylum Firmicutes. The genus Bacteroides still
contains organisms
for
which the taxonomic position remains uncertain; for example, further studies
are needed
for B.
ureolyticus, which is an asaccharolytic, formate- and fumarate-requiring
species, i.e.,
phenotypically
close to Campylobacter gracilis(205). A
novel genus, distantly relatedto the
genus
Bacteroides, contains one species isolated from a human brain abscess, Phocaeicola
abscessus,
which is asaccharolytic and motile (4).
The
genus Alistipes, which belongs to the family Rikenellaceae, currently
includes five
species,
A. finegoldii, A. indistinctus, A. onderdonkii, A. putredinis, and A.
shahii
(136, 157, 191), which are anaerobic, nonmotile, and, except for coccoid-shaped A.
indistinctus
cells, straight or slightly curved rods. The type species of the
genus,A.
putredinis
(formerly Bacteroides putredinis), is asaccharolytic,
nonpigmented, and bile
sensitive.
A. indistinctus is also nonpigmented and bile sensitive but it is
saccharolytic,
whereas
the other Alistipes species are saccharolytic, pigmented, and bile
resistant.
The
family Porphyromonadaceae includes five genera with species detected in
humans:
Barnesiella, Odoribacter,Parabacteroides, Porphyromonas, and Tannerella.
Of these
genera,
Porphyromonas is clinically most relevant. There are currently 17
validly
published
Porphyromonas species, and many of them are of animal origin. P.
asaccharolytica,
P. bennonis, P. catoniae, P. endodontalis, P. gingivalis, P. somerae, and P.
uenonis
are rather frequently detected in humans (56, 101, 102, 177, 195, 196).
Most
species
are asaccharolytic and, except forP. catoniae, pigmented, and they are
generally
considered
pathogens. The genus Parabacteroides, consisting ofP. distasonis, P.
goldsteinii,
P.
gordonii, P. johnsonii, and P. merdae (167, 168, 171), is
phylogenetically closely related
to
the genera Tannerella (166) and Barnesiella (169). Parabacteroides
organisms are
saccharolytic
and resistant to 20% bile. The genus Tannerella contains only one
species, T.
forsythia
(formerly T. forsythensis) (166), which
is an important oral pathogen, and the
genus
Barnesiella contains one human species, B. intestinihominis, isolated
from feces (129).
Both
of these species are anaerobic, nonpigmented, nonmotile, pleomorphic rods. The
new
genus
Odoribacter includes two human-derived species; O.
splanchnicus
(formerlyBacteroides) is saccharolytic and bile resistant,
while the newly
described
O. laneus (136) is asaccharolytic and susceptible to 20% bile.
The
family Prevotellaceae includes saccharolytic or moderately saccharolytic
short rods that
produce
acetic and succinic acids as their major end products of fermentation (179).
This
family
has been confronted by a tremendously increasing number of species in the past
few
years.
Most novel Prevotella species (Table 1)
have been isolated from the oral cavity (43–
45, 47, 172)
but also from feces (75) and other sites of the body, where they have been
associated
with various clinical conditions (2, 42, 60, 109, 170).
Two species, P.
heparinolytica
and P. zoogleoformans, are only loosely connected to other Prevotella
species
and,
instead, they cluster phylogenetically with Bacteroides species (146),
while P.
tannerae,
together with some as-yet-uncultivable taxa, appears to represent
a novel genus,
related
to but distinct from Prevotella. Recently, a novel genus, Paraprevotella,
consisting of
two
new species (Table 1) from human feces, was described (130).
In
the family Fusobacteriaceae of the phylum Fusobacteria, the
genera of clinical interest
are Fusobacterium,
Leptotrichia, and Sneathia.These organisms are nonmotile,
pleomorphic
rods,
mainly isolated from the oral cavity. In a study using sequencing of the
16S-23S rRNA
gene
internal transcribed spacer regions ofFusobacterium species (35),
three phylogenetic
clusters
were formed: the first cluster included F. mortiferum, F. varium, and F.
ulcerans; the
second
cluster contained F. nucleatum subspecies, F. naviforme (note
that there are
considerable
inconsistencies in the phenotypic and genotypic characteristics between F.
naviforme
strains obtained by different laboratories; the strain used in
this study by Conrads
et
al. [35] fits with the original description of the species), F. simiae, and
F.
periodonticum;
the third cluster included F. necrophorumsubspecies and F.
gonidiaformans. F.
russii and F. perfoetens formed separate branches. The somewhat
fuzzy
phylogeny
of fusobacteria and wide heterogeneity of F. nucleatum, in particular,
have been
recently
explained by potential horizontal gene transfer that occurred in the close
interaction
of
oral bacteria within dental biofilms (123).
The genus Leptotrichia consists of nonmotile,
highly
saccharolytic, long rods that typically produce lactic acid. Currently, there
are six
validly
described Leptotrichia species: L. buccalis, L. goodfellowii, L.
hofstadii, L. shahii, L.
trevisanii,
and L. wadei (50, 51),
while a former species, L. sanguinegens, isolated from
blood,
was moved to a novel genus, Sneathia (34). “L.
amnionii” (180) is not validly
published
and, in fact, phylogenetically clusters closer to Sneathia sanguinegens than
to
other
Leptotrichia species (50).
Recently,
a novel phylum named Synergistetes was proposed by Jumas-Bilak et al. (92).
So
far,
only two cultivable genera, including obligately anaerobic, nonmotile,
gram-negative
species,
Jonquetella anthropi (91) and Pyramidobacter
piscolens (46), have been isolated
from
humans. However, fluorescent in situ hybridization analysis has revealed that
the oral
cavity
harbors a diverse population of unculturable Synergistetes, which are
large curved
bacilli
(206).
In
the gram-positive phylum Firmicutes, the family Veillonellaceae includes
some clinically
important
genera, which have traditional gram-negative cell walls. The
genus
Dialister includes five species of human origin: D. invisus (41)
and D.
pneumosintes
(formerly Bacteroides pneumosintes) isolated from the oral
cavity, D.
micraerophilus
and D. propionicifaciens (90)
from clinical specimens, and a novel species, D.
succinatiphilus,
from feces (129). These anaero bic or microaerobic, gram-negative
coccobacilli
are asaccharolytic and largely unreactive in biochemical tests (90).
The two
human
species of the genus Megamonas, M. hyper-megale and the novel M.
funiformis
(173), are anaerobic, gram-negative, very large rods. In the genus Selenomonas,
S.
sputigena, S. artemidis, S. dianae, S. flueggei, S. infelix, and S.
noxia, and also the
closely
related Centipeda periodontii, have been isolated from the human oral
cavity (126).
They
are anaerobic, gram-negative, curved motile rods.
Various
families in the phylum Proteobacteria include genera and species with
clinical
importance
in humans. The genera Sutterella and Parasutterella in the
family
Alcaligenaceae consist of asaccharolytic, bile-resistant, gram-negative
short rods, of
which
S. wadsworthensis and the novel S. parvirubra (173)
and P. excrementihominis (135)
have
been isolated from human specimens. The family Desulfovibrionaceae contains
two
genera
of clinical interest, Bilophila and Desulfovibrio. Bilophila
wadsworthia is an anaerobic,
asaccharolytic,
bile-resistant, gram-negative rod and is a significant pathogen in humans
(11).
Of the more than 30Desulfovibrio species, some infrequently cause a
variety of human
infections
(64). In the familyDesulfomicrobiaceae, the genus Desulfomicrobium
includes one
human
species, D. orale, which is associated with periodontal diseases (106).
The
genus
Anaerobiospirillum of the family Succinivibrionaceae includes two
species, A.
succiniciproducens
and A. thomasii , isolated from feces of humans, cats, and
dogs
(121).Anaerobiospirillum,
Desulfovibrio, and Desulfomicrobium organisms are strictly
anaerobic,
gram-negative, motile, spiral-shaped bacteria that reduce sulfate.
Complete
genome sequences are available for the following members of this
group:
Bacteroides thetaiotaomicron strain VPI-5482 (accession no. NC_004663)
(215); Bacteroides
fragilis NCTC 9343 (NC_003228) (27); B.
fragilis YCH46 (NC_006347)
(105); Bacteroides
vulgatus ATCC 8482 (NC_009614) (216);Parabacteroides
distasonis ATCC
8503
(NC_009615) (216); Porphyromonas gingivalis W83 (NC_002950)
(140);Porphyromonas
gingivalis ATCC 33277 (NC_010729) (138); Desulfovibrio
desulfuricans
G20 (NC_007519) (unpublished); D. desulfuricans 27774
(NC_011883)
(unpublished);
Desulfovibrio magneticus RS-1 (NC_012796) (139); Desulfovibrio
salexigens
DSM 2638 (NC_012881) (unpublished); Desulfovibrio vulgaris DP4
(NC_008751)
(207); D.
vulgaris “Hilden (NC_002937) (79); D.
vulgaris “Miyazaki” (NC_011769)
(unpublished);
Fusobacterium nucleatum ATCC 25586 (NC_003454) (93); Leptotrichia
buccalis
C-1013-b (NC_013192) (83).
Interestingly, the genome sequence for F.
nucleatum
revealed that although this species has a gram-negative cell wall,
including an
outer
membrane, a significant proportion of genes were related to homologues from
grampositive
species
in the phylum Firmicutes, suggesting that Fusobacterium has a
grampositive
evolutionary
history (93).
Current
methods used for taxonomic studies are mainly based on nucleic acid analyses,
in
particular,
sequencing of the 16S rRNA gene and comparison of these sequences, in order to
reveal
the phylogenetic relatedness of taxa. This approach does not necessarily
correlate
with
phenotypic characteristics, such as cell and colony morphologies, atmospheric
growth
requirements,
and various biochemical test results, which are still widely used in clinical
microbiology
laboratories. However, appropriate atmospheric requirements should be
determined
for all isolates, because the true anaerobes can be differentiated from
facultatively
anaerobic bacteria by their inability to grow in the presence of oxygen and by
their
susceptibility to metronidazole (89).Table
2 presents differential characteristics of
clinically
relevant genera within the gram-negative anaerobic rods.
Back
to top
TABLE 2 - Characteristics of genera representing
gram-negative anaerobic rods
isolated from clinical specimensa
EPIDEMIOLOGY AND TRANSMISSION Back
to top
Gram-negative
anaerobic rods inhabit the mucosal surfaces of the oral cavity and
gastrointestinal
tract of animals and humans. In fact, some of these organisms, such
as Fusobacterium
and Prevotella, are ubiquitous members of the mouth from the early
months
of life (102) and, when teeth erupt, are an integral part of dental biofilms
(99).Recently,
Prevotella has been shown among the dominant genera in other habitats,
such
as the esophagus and stomach (13, 149),
which have previously been considered to
have
a very limited microbial diversity. In the gut, Bacteroides becomes part
of
themicrobiota
in early infancy, although as a result of cesarean section, the colonization of
the B.
fragilis group organisms can be delayed and the levels are greatly reduced
(150). Two
bacterial
phyla, Firmicutes and Bacteroidetes, dominate in the gut, and
changes in their
relative
proportion seem to have an impact on host physiology, with the proportion
of Bacteroidetes
being decreased in obese people (113, 212).
In the female genital tract,
when
the vaginal hydrogen peroxide-producing lactobacilli decrease in
numbers,
Prevotella species increase and become an important part of the
microbiota with
other
commensal vaginosisassociated microorganisms (186).
CLINICAL SIGNIFICANCE Back to
top
Anaerobes,
originating mainly from the indigenous microbiota, are detected typically in
polymicrobial
infections associated with mucosal surfaces close to the site where they
reside.
Most
infections are acquired when the integrity of the colonized mucosa or lumen is
breached
by
trauma, underlying disease, or during surgery. Exceptions to this endogenous
acquisition
include
clenched-fist wounds and animal and human bite wounds. Gram-negative anaerobes,
such
as B. fragilis, are involved in a variety of infections associated with
considerable
morbidity
and mortality (212). Table 3 summarizes the infectious sites where gram-negative
anaerobic
organisms have been frequently isolated from clinical specimens. Anaerobic
bacteria
can occasionally spread to adjacent tissues and the bloodstream with serious
consequences.
Localized dentoalveolar infections can result in life-threatening spread of
oral
anaerobes
along tissue spaces of the head and neck up to the mediastinum (55).
In cases
when
gram-negative anaerobes gain entrance to the bloodstream and trigger a systemic
inflammatory
response, this may result in sepsis or infective endocarditis with a fatal
outcome.
The gastrointestinal tract and the oropharynx are the most common sources for
anaerobic
bacteremias, with gastrointestinal surgery and underlying malignancies being
the
major
predisposing factors (17, 108). In the oral cavity, inflamed periodontal tissues offer an
open
portal for a myriad of oral anaerobes to the circulation via the daily
practices of oral
hygiene
and chewing food (7, 116). This calls attention to the importance of prevention of
oral infections, especially
in patients at increased risk for infective endocarditis.
Bacteroides
and Related Genera
Among
the anaerobes in clinical specimens, members of the bile-resistant B.
fragilis group
are
the most commonly encountered and are more virulent and resistant to
antimicrobial
agents
than most other anaerobes. Although other intestinal Bacteroides species
outnumber
B. fragilis 10- to 100-fold, B. fragilis proved to be the most
frequent
Bacteroides found in specimens from blood, ulcers, abscesses, bronchial
secretion,
bone,
intra-abdominal infections, inflamed appendix, and the head (212).
In the Wadsworth
Anaerobe
Collection database, consisting of more than 3,000 clinical specimens, B.
thetaiotaomicron
and B. ovatus, as well as B. capillosus (currently
known as
a Pseudoflavonifractor
species), in descending order, were also detected in these specimens,
but
less frequently. Another U.S. study that included over 5,000 isolates of theB.
fragilis
group from clinical specimens confirmed this: B. fragilis was
most common (52%),
followed
by B. thetaiotaomicron (19%) and B. ovatus (10%) (185).
In children, B. fragilis is
the
main anaerobic organism recovered from intra-abdominal infections (114, 212);
for
instance,
it is isolated from nearly all tissue specimens of acute appendicitis (156).
Around
10
to 20% of the B. fragilis strains are able to produce enterotoxin; these
strains have been
associated
with diarrhea and, in addition, their proportion seems to be higher than that
of
nontoxigenic
strains among blood culture isolates (175).
The B. fragilis group
organisms(here
also including Parabacteroides distasonis) and other Bacteroides species
are
the
most frequently isolated pathogens from bloodstream infections with involvement
of
anaerobes
(17, 108, 181, 212). Recently, the first recoveries of two
novel
Bacteroides species, B. dorei and B. finegoldii, were reported
from blood
(181). Bacteroides
involvement in infective endocarditis, where B. fragilis of
gastrointestinal
origin
is the most frequent finding, tends to be more serious than that of other
anaerobes,
sometimes
with a fatal outcome (14, 23). The B. fragilis group has been recovered from
pericarditis
samples (24) and, often as a single isolate, from septic arthritis as well as
from
osteomyelitis
samples (22). Although spondylodiscitis caused by anaerobes is not common,
the
involvement of B. fragilis needs to be taken into account in cases with
potential
bacteremia
of intestinal origin (48). By hematogeneous spread, the B. fragilis group and
otherBacteroides
organisms can reach the brain, causing abscesses (111, 154, 194).
In
addition,
members of this group are among the predominant isolates from burn wound
infections,
with potential involvement in sepsis in this context (133). Bacteroides
species can
be
involved in part in polymicrobial necrotizing soft tissue infections (212),
and the B.
fragilis
group is, after gram-positive anaerobic cocci, one of the most
common anaerobic
findings
in infected moderate to severe diabetic foot wounds (32, 141).
In cat and dog bite
infections,
B. tectum was the most common Bacteroides isolate (197).
Pigmented,
bile-resistant Alistipes species, A. finegoldii, A. onderdonkii, and
A. shahii, have
been
strongly connected to appendicitis, both in children and in adults (156, 191).
In
addition,
A. finegoldii has been isolated from blood, A. shahii from
intra-abdominal fluid,
and A.
onderdonkii from intra-abdominal abscesses and urine (54, 181, 191).
Porphyromonas
and Related Genera
Pathogenic
potential varies between different Porphyromonas species. Of the three
oral
Porphyromonasspecies, P. endodontalis and P. gingivalis are
known significant
pathogens.The
detection rate of P. gingivalis,one of the major periodontal pathogens,
increases
with age (80, 103). In addition to periodontitis, it has been frequently detected
in
oral
specimens from necrotizing ulcerative gingivitis, infected root canals, peri-
implant
lesions,
and acute apical abscesses (62, 67, 155, 183).
Besides the oral cavity, it has been
detected
in clinical specimens from various body sites, e.g., intra-abdominal sites (122),
vaginal
samples in women with bacterial vaginosis (151),
amniotic fluid (112), synovial
specimens
of rheumatoid arthritis and psoriatic arthritis patients (124)
and, together with
some
other periodontal organisms, in occluded arteries of lower extremities of
Buerger’s
disease
patients (84). P. endodontalis is one of the dominant organisms in
infected root
canals
and in acute dental abscesses (67, 164, 183)
but may also be involved in chronic
periodontitis
(104). P. uenonis,which is phenotypically similar to P.
endodontalis and P.
asaccharolytica,
has been detected in polymicrobial infections below the waistline:
appendicitis,
peritonitis, pilonidal abscess, an infected sacral decubitus ulcer, and
bacterial
vaginosis
(56, 57). P. asaccharolytica and P. somerae were among the
anaerobic isolates
from
moderate to severe diabetic foot infections (32).
At the VA Wadsworth Medical Center
laboratory,
the 58P.somerae isolates originated from a variety of specimens,
including lower
extremity
skin and soft tissue or bone, in particular, and inguinal or sacral area
abscess,
intra-abdominal
abscess, transtracheal aspirate, axillary abscess, mastoiditis, blood culture,
brain
tissue, and infected scalp (presented in order of their frequency) (195). A
novel
Porphyromonas species, P. bennonis, has been detected in human
wound infections
and
abscesses, especially in patients with chronic skin and soft tissue lesions in
the
perirectal,
buttock, and groin regions (196). Although P. catoniae inhabits
the oral cavity
without
any disease association described to date, it has been isolated from an
abdominal
abscess
(101). P. gingivalis and Porphyromonas species of animal
origin, e.g., P.
cangingivalis,
P. canoris, P. cansulci, and P. macacae, have been
encountered in humans
with
animal bite infections (197).
Parabacteroides
species are common inhabitants of the human gut, and P.
distasonis is one
of
the anaerobes of clinical importance in specimens from intra-abdominal
infections and
inflamed
appendixes, where P. goldsteiniiand P. merdae can also be found (189, 212).
In
addition,
P. distasonis, P. merdae, and a novel species, P. gordonii, have
been isolated from
human
blood (85, 171, 181, 212).
Tannerella
forsythia is considered one of the major periodontal pathogens (80, 200).
In
addition,
it is one of the predominant organisms in root canal infections (164)
and has been
found
in infected sites around dental implants (155).
This oral bacterium has been recovered
from
vaginal samples in women with bacterial vaginosis (151) as
well as from synovial
specimens
of rheumatoid arthritis and psoriatic arthritis patients (124).
Prevotella
and Related Genera
Prevotella
species are among the dominating microorganisms of the oral
cavity, where they,
despite
their commensalism, can be involved in nearly all types of oral infections.
Interestingly,
P. melaninogenica, which is a common anaerobic organism in saliva
(100, 102),
is also one of the most prevalent anaerobes, together withF. nucleatum, P.
intermedia,
and P. buccae, in infected human bite lesions (198).
Although the cariogenic
microbiota
mainly consists of gram-positive species, some proteolytic gram-negative taxa,
including
P. denticolaand P. tannerae, can be frequently encountered in
advanced carious
lesions
(29). P. intermedia (sensu stricto) is strongly linked to periodontitis
(103, 148),
and P.
intermedia and/or the phenotypically identical P. nigrescenshas been
detected in
samples
from pregnancy gingivitis, necrotizing ulcerative gingivitis, pericoronitis,
periimplantitis,
root
canal infections, and dentoalveolar abscesses
(62, 67, 69, 122, 155, 183, 184).
Also, P. baroniae, a recently described Prevotella species,
proved
to be common in root canal infections and acute dental abscess aspirates (164, 183).
In
noma (cancrum oris) lesions,P. intermedia (sensu lato) is considered a
key organism (49).
In
spreading odontogenic infections, members of the genus Prevotella seem
to play an
important
role, with P. buccae and P. oris the most prominent findings in
this context
(55, 159).
Recently, a new concept of the polymicrobial bacteriology of cystic fibrosis
has
been
presented and, indeed, not only aerobicPseudomonas aeruginosa and Staphylococcus
aureus
but also some anaerobes, especially Prevotella, such as P.
melaninogenica, P.
denticola,
P. oris, and P. salivae, have been detected as one of the
persistently dominating
organisms
in the sputum of these patients (16).
Also, Prevotella taxa were recovered from
bronchoalveolar
lavage fluid of ventilator-associated pneumonia (6).
Furthermore,
various
Prevotella species are found in extraoral infections and abscesses in a
wide range of
body
sites, for instance, P. amnii, P. bivia, P. corporis, P. disiens, P.
intermedia, and P.
nigrescens
in the female genital tract (21, 109, 148, 151); P.
buccalis in urine (40);P.
bergensis,
P. bivia, and P. melaninogenica in infectious lesions of the skin and
soft tissues,
including
diabetic foot lesions (32, 42); P. intermedia and P. nigrescens in
intra-abdominal
and
soft tissue abscesses (122); P. intermedia (sensu lato) and P. melaninogenica in
peritonsillar
and retropharyngeal abscesses (21); P. timonensis in
breast abscesses (60); P.
intermedia
in synovial fluid of arthritis patients (124);
and P. intermediaand P.
nigrescens
(with some other periodontal bacteria) in occluded arteries of lower
extremities of
Buerger’s
disease patients (84). Several Prevotella species, such as P. bivia, P.
buccae, P.
denticola,
P. disiens, and P. nigrescens, have been among anaerobic findings in
bloodstream
infections
(122, 181). P. heparinolytica is a relatively common anaerobic
isolate from animal
bite
wounds (197).
Fusobacterium
and Related Genera
Clinically,
the most important Fusobacterium species are F. nucleatum and F.
necrophorum
(19, 20, 31, 160). F. nucleatum is an oral species which has been divided
into
several
subspecies with variable pathogenic potentials (19).
It is a key organism in
maturation
of pathogenic biofilms in periodontal pockets (187)
and considered an important
pathogen
in peri-implantitis, root canal infections, dentoalveolar abscesses, and
spreading
odontogenic
infections (155, 159, 164, 183). It is also an important etiologic agent in
extraoral
infections and abscesses at a wide range of body sites, being detected from
blood,
brain,
chest, heart, lung, liver, appendix, joint, abdomen, genitourinary tract, and
fetal
membranes
as well as infected human bite lesions
(14, 22, 24, 31, 39, 71, 73, 82, 111, 156, 181, 198).
In a tertiary care hospital where all
episodes
of documented brain abscess cases between 1991 and 2000 were reviewed, in 40%
of
cerebral puncture specimens, only anaerobes were found; of these, F.
nucleatum proved
to
be the most frequently isolated organism, found in one of three of the patients
diagnosed
(111).
The presence of a novel adhesin,Fusobacterium adhesin A, seems to allow
separation
of
oral fusobacteria, F. nucleatum, F. periodonticum, and F. simiae, from
nonoral
fusobacteria,
including F. gonidiaformans, F. mortiferum, F. naviforme, F. russii, and
F.
ulcerans
(72). Because the adhesin was present in F. nucleatum isolated
from intrauterine
infections
but absent among the vaginal species F. gonidiaformans and F.
naviforme, it was
hypothesized
that intrauterine F. nucleatum originates from the oral cavity rather
than the
vaginal
tract. Of the two F. necrophorum subspecies,F.
necrophorum
subsp. funduliforme (biovar B) is a human pathogen (20, 160). F.
necrophorum
is best known for its connection to Lemierre’s syndrome
(necrobacillosis),
which
can be considered an invasive F. necrophorum disease, often with
pleuropulmonar
involvement
(20, 70, 160). It is notable that invasive disease with F. necrophorum may
be
on
the increase (20). In Denmark, the overall mortality of Lemierre’s syndrome,
originating
mainly
from oropharyngeal sites, was reported to be 9% in adolescents, and that of
disseminatedF.
necrophorum infections, originating from lower parts of the body, was 26%
in
elderly
with predisposing diseases (70). In the latter type of
cases, underlying cancers
should
be considered. Interestingly, F. necrophorum can be found in adolescents
as the
causative
agent in approximately 10% of cases of tonsillitis (persistent sore throat) not
caused
by group A streptococci (5, 20, 160). In a Danish retrospective study of 847 patients
with
peritonsillar abscesses from 2001 to 2006, F. necrophorum was detected
in 23% of the
pus
aspirate or swab specimens, most of them growing as a pure culture (97).
Recently,
Riordan
(160), in his extensive review on F. necrophorum, presented a
wide variety of
infections
caused by this organism: in the head and neck, infections included tonsillitis,
peritonsillar
abscess, deep neck space infection, mediastinitis, otogenic infection,
mastoiditis,
sinusitis,
and odontogenic infection; as intracranial complications, infections included
sinus
thrombosis,
cerebral abscess, and meningitis; systemic manifestations included bacteremia,
septicemia,
pleuropulmonary infections, bone and joint infections, soft tissue infections,
intra-abdominal
sepsis, endocarditis, and pericarditis. Other Fusobacterium taxa, such
as F.
necrogenes,
F. ulcerans, and the F. mortiferum-F. varium group, have been
only occasionally
isolated
from human clinical specimens (31, 32, 110,115). F.
nucleatum, an F. nucleatumlike
species
of animal origin, F. canifelinum, and F. russii are of importance
in cat and dog
bite
wounds (36, 197).
Commensal
Leptotrichia species have been implicated in anaerobic bacteremias
inimmunocompromised
patients with lesions of the oral mucosa but are also occasionally
considered
etiologic agents of bacteremia and infective endocarditis in immunocompetent
individuals
(17, 25, 51, 201, 203). L. buccalis, L. goodfellowii, L. trevisanii, and L.
wadei are
the
main Leptotrichia findings in blood specimens. In addition, L. wadei was
reported as a
causative
organism of a severe pneumonia in an immunocompetent subject (95).
In the
female
genital tract, some infectious cases, including intrauterine fetal demise and
septic
abortion,
have been reported in connection with L. amnionii (18, 48, 167).
However, L.
amnionii
was recently isolated from a knee joint specimen of a male patient
(68), i.e., the
case
was unrelated to either the female genital tract or delivery. Based on the high
number
of L.
amnionii bacteria in urine samples from renal transplant recipients, it may
be
considered
one of the etiologic agents of urinary tract infections (38).
In addition to L.
amnionii,
Sneathia(formerly Leptotrichia) sanguinegens, which was
originally isolated from
blood
(32), has been reported from various infectious conditions of the
female genital tract,
such
as preterm labor, peripartum bacteremia, and pyosalpinx, and in amniotic fluid
in
association
with various clinical syndromes (36, 37, 48). Leptotrichia
andSneathia are among
the
taxa of which concentrations have been suggested for use as potential markers
of
bacterial
vaginosis and its treatment response (58).
Moreover, these taxa are considered
clinically
relevant intra-amniotic pathogens (37, 70)
and, in fact, it has been suggested that
the
role of L. amnionii and S. sanguinegens in preterm labor is
currently underestimated.
Other Gram-Negative Anaerobic Rods
In
the novel phylum Synergistetes, there are two cultivable species
representing two genera,
i.e.,
Jonquetella anthropi and Pyramidobacter piscolens, which both
have been isolated from
human
clinical specimens. The J. anthropi isolates came from breast abscess,
pelvic abscess,
sebaceous
cyst, wound, and peritoneal fluid specimens (91),
while P. piscolens isolates were
from
odontogenic abscesses and the periodontal pocket and gingival crevice (46).
Two Dialister
species, D. pneumosintes and D. invisus, are inhabitants of
the oral cavity,
where
they have been implicated as pathogens in endodontal and periodontal infections
as
well
as acute dental abscesses (37,41, 147, 163, 183).
In addition, they have been
recovered
from infectious specimens from sites such as blood, brain abscesses,
bronchoalveolar
lavage fluids from patients with ventilator-associated pneumonia or cystic
fibrosis,
urine, and human bite wounds (6, 7, 16, 40, 128, 165, 198). A
considerable portion
of D.
pneumosintes isolates originate from various cutaneous or soft tissue
infections (128).
It
may be that a patient’s poor dental hygiene is a predisposing factor in certain
nonoral
cases,
e.g., bacteremia and brain abscess (6, 165).D.
micraerophilus, which is not a strict
anaerobe,
and D. propionicifaciens have been isolated from a variety of clinical
specimens,
mainly
below the waistline (90, 128).
Selenomonas
species and Centipeda periodontii are motile organisms
recovered from the
oral
cavity, where they are components of dental biofilms (98, 102). S.
sputigena has been
found
in specimens from necrotizing ulcerative gingivitis, generalized aggressive
periodontitis,
and acute dental abscesses (52, 62, 183), S.
noxiahas been reported in chronic
and
aggressive periodontitis lesions (52, 199),
and C. periodontii has been isolated from
endodontic
infections (182). Furthermore, there have been a few reports on the involvement
ofSelenomonas
in nonoral infections, including cystic fibrosis and bacteremia (15, 16, 88).
S.
wadsworthensis, a microaerobic organism phenotypically but not phylogenetically
close to
fastidiousCampylobacter
species, has been isolated from a variety of infections, such as
appendicitis,
peritonitis, and rectal or perirectal abscesses (125, 211).
B.
wadsworthia is a significant human pathogen in polymicrobial intra-abdominal
infections,
especially
in appendicitis, at an increasing frequency with deteriorating disease status (11).
In
gangrenous appendixes of children, B. wadsworthia is even more common
than in those of
adults
(156). Moreover, B. wadsworthia has been isolated from
abscesses at various body
sites
as well as from blood (11, 204).
Some
Anaerobiospirillum and Desulfovibrio organisms reside in the
gastrointestinal tract of
humans
but can also be infrequently encountered in clinical specimens; typical cases
are
bacteremia
and abdominal infections in immunocompromised patients (64, 88, 120, 152).
Of
the
two Anaerobiospirillum species, A. succiniciproducenshas been
connected to bacteremia
and
diarrheal illness, whereas A. thomasii is considered a potential cause
of diarrhea but not
of
bacteremia (121). In the case of diarrhea, the possibility of zoonotic
transmission exists
(120).
The main Desulfovibrio species isolated from human infections include D.
desulfuricans,
D. fairfieldensis, D. piger, and D. vulgaris (64, 88, 118, 153, 209). D.
desulfuricans
is common also in the environment, whereas D. fairfieldensis and
D. piger have
been
detected only in humans so far. D. fairfieldensis, which may have a
higher pathogenic
potential
than the other species, has been isolated from blood, in particular, but also
from
various
intra-abdominal sites, urine, and periodontal pockets (153).
In addition to D.
fairfieldensis,
an oralDesulfomicrobium species, D. orale, has been
connected to periodontitis
(106).
COLLECTION, TRANSPORT, AND STORAGE OF
SPECIMENS Back to top
General
guidelines for collection, transport, and storage of specimens are discussed
in chapters
9 and 16 of this Manual. Specimens suitable for the isolation of
anaerobes include
aseptically
obtained blood, tissue biopsies, aspirates (e.g., cerebrospinal fluid, joint
fluids,
and
pus), root canal exudates, and subgingival plaque. Appropriate respiratory
tract
specimens
include bronchoscopic protected bronchoalveolar lavage fluid, and expectorated
sputum
samples may be collected from patients with cystic fibrosis (202).
Also, wound and
ulcer
specimens should preferably be taken by tissue biopsy, wound curettage, or
aspiration.
For
example, for diabetic foot ulcers, the lesion is cleaned and carefully
debrided, and tissue
samples
are collected from the base or progressive edge, where bacteria actively
multiply
(32).
Infected tissue, obtained by excision or biopsy, is always preferable to pus as
a clinical
specimen.
However, when pus is collected, it is best aspirated into a syringe through a
needle
and injected into an anaerobic transport vial containing an oxidation-reduction
indicator
(e.g., products from Anaerobe Systems, Morgan Hill, CA, and Becton Dickinson,
Sparks,
MD). It is notable that syringes used for aspiration should not be used as
transporters
because of the potential danger of needle stick injuries or accidental
expulsion
and
because oxygen diffuses through plastic syringes (89).
Mucosal or cutaneous swabs are
not
recommended. In cases where bacteremia is suspected, a 20-ml volume of blood
(at
least
two separate samples) is recommended for cultures. Anaerobic culture is
important,
especially
in patients with complex underlying diseases and when the source of bacteremia
is
unknown
(85,108). Also, blood collected from patients with abdominal or gynecological
processes,
peritoneal abscess, dirty wound, decubitus ulcers, osteomyelitis, or spreading
oropharyngeal
disease should be examined for anaerobes.
Specimens
must be transported to the laboratory under anaerobic conditions without delay
for
further processing. An optimal transport system is one that is able to maintain
the
viability
of anaerobic organisms without allowing the overgrowth of aerobic bacteria.
However,
if clinical specimens contain fastidious organisms, the transport to the
clinical
laboratory
should occur within 24 h (30). Tissue samples are best
transported in specific
anaerobic
transport vials or in loosely capped containers sealed in gas-impermeable bags
in
which
an anaerobic atmosphere has been generated. For small tissue and biopsy
specimens
and
for subgingival and root canal samples, a semisolid anaerobic transport medium
(e.g.,
those
of Anaerobe Systems and Becton Dickinson), in which the specimen can be
submerged,
can be used.
Further
guidance for the collection of specimens from different body sites and by
various
methods
as well as transport systems and anaerobic techniques can be found in more
detail
elsewhere
(89).
For
long-term storage, 2- to 3-day-old cultures can be transferred into vials
containing
sterilized
20% skim milk and kept frozen at −70°C.
DIRECT EXAMINATION Back
to top
The
gross appearance, fluorescence under long-wave UV light, and odor of the
specimen can
give
the laboratory valuable clues to the presence of anaerobes. A fetid or putrid
odor due to
volatile
short-chain fatty acids and amines is always associated with the presence of
anaerobes
in the sample. Black necrotic tissue and/or red fluorescence of the sample may
be
indicative
of the presence of pigmented gram-negative rods (89).
The
Gram stain is still the fastest, simplest, and most likely to yield significant
information
and
should be prepared from all specimens accepted for anaerobic culture. Many
gramnegative
anaerobic
rods, e.g., different species within the genus Fusobacterium, have
unique
cell
morphology. For instance, a Gram-stained smear with highly pleomorphic
gram-negative
rods
in blood culture from a septic patient following a sore throat may indicate
invasive F.
necrophorum
infection (20). The morphotypes and relative quantities of the bacteria and
host
inflammatory cells present in the preparation should be reported. Gram stain
using the
Nugent
criteria for interpretation of vaginal discharge is still considered the best
method for
diagnosis
of bacterial vaginosis (107).
Molecular Detection
Molecular
methods are increasingly used for direct detection of bacteria from clinical
specimens.
As yet, they are not widely used for routine diagnostics but are mainly used in
specialized
oral and other research microbiology laboratories.
For
the detection of fastidious organisms and potential pathogens, sequencing of
the 16S
rRNA
gene has been successfully used not only for bacterial identification in
typical
polymicrobial
lesions, such as periodontitis, endodontic infections, and spreading
odontogenic
infections
(104, 147, 159, 183, 206), but also in infections where the involvement of
anaerobic
bacteria has not been traditionally taken into account, e.g., cystic fibrosis (16).
It
is
notable that a culture-based approach can underestimate the presence of
etiologic but
fastidious
or uncultivable organisms in clinical specimens. For example, Prevotella species
are
highly prevalent in the lungs of cystic fibrosis patients (16, 202),
but routine culture of
sputum
does not include anaerobes and potential anaerobic pathogens are not reported.
Furthermore,
in women with preterm labor whose amniotic fluid tested positive by culture or
PCR,
seven species, including S. sanguinegens and L. amnionii, were
detected by PCR only
(39).
In addition, in a study of urinary tract specimens from renal transplant
recipients (40),
the
16S rRNA PCR method detected a wide range of bacteria, including gram-negative
anaerobes,
such as B. vulgatus, D. invisus, F. nucleatum, L. amnionii, P. buccalis, and
P.
ruminicola,
in culture-negative samples.
A
checkerboard DNA-DNA hybridization method, in which a set of DNA samples are
hybridized
against large numbers of DNA probes on a single support membrane, was first
developed
to investigate the presence of DNA of target organisms (up to 40 species at a
time)
in dental plaque (187). Recently, it has been used for other clinical specimens, such
as
serum
and synovial fluid of patients with active arthritis (124)
and vaginal samples for
detecting
selected target microbes in bacterial vaginosis (151).
ISOLATION PROCEDURES Back
to top
Except
for blood and joint fluid cultures, the use of liquid medium as the only
anaerobic
culture
technique is not acceptable (89). The use of solid
nonselective medium together with
selective
medium increases the yield and saves time in terms of recognition and isolation
of
colonies.
The selective media are chosen based on the expected micro-biota at the
collection
site,
or in the case of bite wounds, on the oral microbiota of the biter (human or
animal).
Freshly
prepared or prereduced and anaerobically sterilized medium should be used (89).
Different
basal media differ in their abilities to support the growth of anaerobes;
brucella
base
and fastidious anaerobe agar (Lab M, Bury, United Kingdom) may be the best
basal
media
for isolation of gram-negative anaerobic rods. In particular, fastidious
anaerobe agar
enhances
the growth of fusobacteria (20). In academic centers
performing large-scale
anaerobic
bacteriology, it would be ideal to use two different basal media to maximize
isolation
efficiency.
Culture
methods are found in chapter 17. The minimum medium setup for isolating gramnegative
anaerobic
rods includes (i) a nonselective, enriched, brucella base sheep blood agar
plate
supplemented with vitamin K1and hemin (BA); (ii) a kanamycin-vancomycin laked
sheep
blood agar plate for the selection of Bacteroides andPrevotella species;
and (iii)
a Bacteroides
bile-esculin (BBE) agar plate for specimens from areas below the diaphragm
for
the selection and presumptive identification of the B. fragilis group
and Bilophila species.
BBE
and kanamycin-vancomycin laked sheep blood are also available as biplates. A
phenylethyl
alcohol sheep blood agar plate used to prevent overgrowth by aerobic
gramnegative
rods
and swarming of some clostridia is indicated for purulent specimens and in the
case
of mixed infections. Use of a metronidazole disk on nonselective agar is useful
for the
detection
of gram-negative obligate anaerobes; however, it may mask the presence of
infrequently
encountered metronidazole-resistant organisms.
After
inoculation, the anaerobic plates are immediately incubated at 36 to 37°C in an
anaerobic
environment, such as an anaerobic bag, jar, or chamber. Alternatively, setup and
incubation
may all be done in an anaerobic chamber. Plates should not be exposed to air
during
the first 48 h, to avoid loss of the more oxygen-sensitive species. The
availability of
an
anaerobic chamber enables the examination of the culture whenever necessary. An
incubation
period of 48 h will reveal the presence of rapidly growing strains, such
as Bacteroides
or clostridia, but reincubation for 5 to 7 days for primary plates is
recommended,
since some species, such asBilophila, Desulfovibrio, and Porphyromonas,
may
not
be detected with shorter incubation times and require at least 4 to 5 days for
growth
(11, 64, 89).
Increased
awareness of the importance of anaerobic organisms as a cause of systemic
infections
may contribute to the increase of their isolation and detection in blood
samples in
clinical
microbiology laboratories (78). To reliably detect
anaerobic organisms, the LYTIC 10
Anaerobic/F
Bactec medium (Becton Dickinson) has been shown to be a rapid and reliable
method,
improving the detection of low levels of anaerobic bacteria, such
as Prevotella
and Fusobacterium, in the sample (7).
IDENTIFICATION Back to top
After
anaerobic incubation, the relative quantities of distinct colony types are
recorded.
Plates
should be examined with a dissecting microscope to facilitate detection. Even
after
incubation
for 7 days, certain species, such as Desulfovibrio and Dialister, grow
as
transparent
colonies that are pinpoint in size and are easily overlooked in mixed cultures
(64, 90, 118, 153).
The isolates should then be subcultured onto BA and, at this point, a
rabbit
laked blood agar plate for the rapid demonstration of pigment production and an
egg
yolk
agar plate for the demonstration of lipase, lecithinase, and proteolytic
activities may
also
be inoculated. The primary plates are reincubated along with the purity and
test plates.
Presumptive Identification
Colony
morphology of an isolate in pure culture can be useful for presumptive
identification.
For
example, F. nucleatum can appear on the plate as speckled, iridescent,or
bread crumblike
colonies,
while B. wadsworthensis has typical black-centered colonies on BBE (89).
Colony
morphology, together with the capability of erythrocyte agglutination, is among
the
features
that can be used to separate the two F. necrophorum subspecies by using
phenotypic
tests (87). The agglutination procedure, using human and chicken
erythrocytes,
is
performed by a glass slide method, where agglutination is observed by mixing a
drop of
bacterial
suspension and a drop of erythrocyte suspension on a microscope slide. F.
necrophorum
subsp.funduliforme (agglutination-negative) colonies are
pulvinate, creamy,
and
glistening with entire margins, whereas F.
necrophorum
subsp. necrophorum (agglutination-positive) colonies are
convex or umbonate,
waxy,
and dull with erose margins. Also, observation of hemolysis may be of
diagnostic
value;
for instance, both F. necrophorum subspecies exhibit beta-hemolysis when
grown on
horse
blood agar (87). Hemolytic properties on human blood may aid in separation of
different
Leptotrichia species (50). Production of pigment is
another visible characteristic
valuable
in presumptive identification; the pigmented gram-negative anaerobic rods are
composed
of saccharolytic and asaccharolytic species of the
genera
Prevotella and Porphyromonas and three pigment-producing Alistipes
species, A.
finegoldii,
A. onderdonkii, and A. shahii. In this context, it is notable that the
statement
“after
4 days incubation on laked rabbit blood agar, colonies appear black” in the
original
description
of A. onderdonkii and A. shahii (191) is
incorrect. Instead, the grade of
pigmenting
is light or moderately brown on rabbit laked blood agar, and pigment also
appears
on BA after extended incubation. The
pigmented
Prevotella and Porphyromonas species vary greatly in the degree
and rapidity of
pigment
production (2 to 21 days), which ranges from buff to tan to black,depending
primarily
on the type of blood and the composition of the base medium used in the agar
(89).
Fluorescence under long-wavelength UV light can be helpful in presumptive
identification;
pigmented Prevotella and Porphyromonas colonies typically
fluoresce red,F.
nucleatum
and F. necrophorum fluoresce yellow-green,
and Desulfovibrio
and Bilophila species, when tested with a drop of NaOH on a swab of
cell
paste,
fluoresce red due to the presence of desulfoviridin pigment (87,89, 209).
Microscopic
determination of the morphology of Gram-stained bacterial cells can aid
presumptive
identification of the organisms present. Among fusiforms, F. nucleatum usually
exhibits
long, spindle-shaped cells with tapered ends, while F. necrophorum and F.
mortiferum
have highly pleomorphic cells, with or without swollen areas and
large round
bodies
(20, 87, 89). Leptotrichia cells, which often stain gram-positive in
fresh cultures, have
been
usually considered long rods; however, this description fits only with L.
buccalis, L.
hofstadii,
L. shahii, and L. trevisanii (50, 201). Dialister
species are small coccobacilli,
making
their separation from gram-negative cocci difficult (90). Desulfovibrio
piger typically
stains
bipolar (209).Wet slide preparation for microscopic examination reveals the
motility of
gram-negative
anaerobes: Selenomonas displays a characteristic tumbling motility,
often
moving
laterally across the field; Anaerobiospirillum cells are spiral with
corkscrew-like
motility;
Desulfovibrio species, except for D. piger, appear as curved rods
with rapid,
progressive
motility (64, 117, 121).
The B.
fragilis group, in particular, and most Bacteroides species are
typically bile resistant
(89, 212).
Pigment- producing Alistipes can be readily distinguished from
pigmented
Porphyromonas and Prevotella species by the resistance to 20% bile
(191).
Among
fusobacteria, F. mortiferum, F. varium, and some strains of F.
necrophorum grow in
the
presence of bile, whereas F. nucleatum does not. The profile of
susceptibility to specialpotency
antimicrobial
disks (a zone size of ≥10 mm is considered susceptible), containing
vancomycin
(5 μg), kanamycin (1,000 μg), and colistin (10 μg), is useful in presumptive
identification
of many gram-negative anaerobic taxa (Table 2).
Gram-negative anaerobic
rods
are typically resistant to vancomycin, with pigmented Porphyromonas species
the only
exception.
Susceptibility to both kanamycin and colistin is characteristic
of Fusobacterium
and Leptotrichia species and S. wadsworthensis. To
differentiate members
of
the genera Dialister and Veillonella, special potency disks can
be helpful; Dialister species
are
resistant to colistin, whereas Veillonella species are usually
susceptible, except for V.
montpellierensis
and V. ratti (90).
Among motile gram-negative
organisms,
Anaerobiospirillum is usually susceptible to colistin, unlike
most
Desulfovibrioand Selenomonas isolates.
In
addition to the characteristics listed above, there are some simple tests that
are within the
scope
of most clinical laboratories. For example, a bile-resistant organism with
typical
darkening
of the center of colonies on BBE can be easily recognized as B. wadsworthia by
its
strong
catalase reaction with 10 to 15% H2O2 (11),
and by combining positive indole and
lipase
reactions, a Fusobacterium-like organism can be tentatively identified
as F.
necrophorum
(20). An indole- and lipase-positive short rod that forms
black-pigmented
colonies
and fluoresces red can be identified as P.intermedia/P. nigrescens, while
P.
pallens
resembles these indole-positive species but has lighter pigment
and is lipase
negative.
Bilophila and Sutterella typically reduce nitrate. A
characteristic smell may guide
the
identification; a foul smell produced by butyric acid and other metabolic
products is
typical
for Fusobacterium species (89),
while a strong sulfur smell is typical for the presence
ofDesulfovibrio
species (64, 209).
Biochemical Testing
In
culture-based biochemical testing, the main techniques for classification of
anaerobic
organisms
and distinction of individual species include sugar fermentation reactions,
using
prereduced,
anaerobically sterilized carbohydrates or commercial test kits, and the
determination
of enzyme profiles with individual diagnostic tablets or preformed enzyme kits.
In
addition, the determination of major volatile fatty acid end products of
glucose
metabolism,
as detected by gas liquid chromatography (GLC), is a useful adjunct to
biochemical
and physiological tests (89), but together with analysis of the long-chain fatty
acids
found in bacterial cell walls it is beyond the scope of most clinical
laboratories.
Commercially
available test kits, such as the API 20A, API ZYM, and Rapid ID 32A
(bioMerieux,
Marcy-l’Etoile, France), RapID ANA II (Remel, Lenexa, KS), BBL crystal
identification
(Becton Dickinson), and AN microplates (Biolog Inc., Hayward, CA) systems,
are
used for testing preformed enzyme and carbohydrate fermentation profiles in
clinical
microbiology
laboratories (66). Diagnostic tablets (e.g., from Rosco, Taastrup, Denmark, and
Key
Scientific, Stamford, TX) are also useful for determining individual enzyme
reactions of
anaerobic
isolates. A heavy inoculum from 2- to 3-day-old cultures should be used for
testing
to
obtain optimal reactions. When using different test systems, variation of test
results is
expected
due to differences in the substrate specificities. These rapid, easy-to-use
systems
are
best suited for fast-growing and biochemically reactive anaerobes, such as the B.
fragilis
group organisms, but even then it may not be possible to reliably
identify the isolate
to
the species level. The Vitek 2ANC card (bioMerieux) is a new automated system
for rapid
identification
of anaerobic bacteria from clinical specimens (132, 158).
According to the
results
of a clinical trial performed in three large tertiary care centers (158),
the Vitek 2 ANC
card
(bioMerieux) proved to be acceptable for routine use in laboratories; however,
the
system
incorrectly identified a considerable number of clinically relevant species,
such as F.
necrophorum,
P. intermedia, and P. melaninogenica, and did not include clinical
isolates that
were
not in the system’s rather limited database. However, skillful reading of the
Gram stain
preparation
considerably improves the percentage of correct identifications (132).
The
most commonly encountered bile-resistant organisms in clinical specimens belong
to
the B.
fragilis group organisms. They grow as gray, circular, convex, and entire
colonies on
BA
and, in addition, grow well on BBE where they (except for B. vulgatus)
blacken the agar
by
hydrolyzing esculin. Based on their resistance to special-potency antibiotic
disks
(vancomycin,
kanamycin, and colistin) and a few rapid tests, such as the catalase, indole,
esculin,
and α-fucosidase tests, they can be initially reported as B. fragilis group
or
organisms
most closely related to this group (89).
The genus Parabacteroides now includes
the
former B. distasonis and B. merdae(167),
and B. slanchnicus has been moved to the
genus
Odoribacter (74). In addition, some newly described, clinically relevant species
include
B. massiliensis, B. nordii, B. salyersiae, P. goldsteinii, and P.
gordonii(53, 171, 188, 189).
All Parabacteroides species are negative for indole and α-
fucosidase,
distinguishing them from the B. fragilis group organisms. P. goldsteinii
is
phylogenetically
and phenotypically very similar to P. merdae; however, P. goldsteinii
is
positive
for α-glucosidase and β-glucosidase (Rosco), whereas P. merdae is not (189).
Furthermore,
the positive b glucuronidase reaction of P. goldsteinii and P. merdae
separates
them
from P. distasonis. Unlike other Parabacteroides species, P.
gordonii does not hydrolyze
esculin
and does not ferment treha-lose (171).
Most of the B. fragilis group and related
organisms
are highly fermentative. Table 4presents the key characteristics for distinguishing
the B. fragilis group
organisms, including Parabacteroidesspecies and O. splanchnicus.
A.
putredinis (formerly Bacteroides putredinis), the type species of the
genus Alistipes, is
nonpigmented,
asaccharolytic, bile sensitive, and positive for indole and catalase (157).
In
contrast,
most Alistipes species can be separated from Bacteroides and A.
putredinis by their
pigment
production, although demonstration of this trait may require prolonged
incubation.
A. finegoldii, A. onderdonkii, and A. shahii strains grow well on
solid media,
where
colonies appear beta-hemolytic, but not in liquid media, with or without
supplements
(191).
They are variably saccharolytic due to their poor growth in liquid media and
are bile
resistant
and catalase negative. Two enzyme reactions may be able to separate
pigmented
Alistipes species: A. finegoldii is positive for α-fucosidase but
negative for β-
glucosidase,
whereas A. onderdonkii is negative and A. shahii is positive for
both enzymes
(191).
Tannerella
forsythia is a fastidious oral pathogen, and human strains require exogenous
Nacetylmuramic
acid
for growth in pure cultures (166, 200).
Key characteristics of T.
forsythia
are its sensitivity to bile and positive trypsin and esculin reactions.
Notably, animal
strains
from bite wound infections are positive for catalase and indole (81).
Porphyromonas
species, except for P. catoniae, produce pigment. The
identification of the
closely
related and phenotypically similar P. asaccharolytica, P. endodontalis, and
P.
uenonis
is difficult due to their slow pigment production and inactivity
in biochemical testing.
Testing
with prereduced, anaerobically sterilized carbohydrates is not helpful in
distinguishing
these species, but glyoxylic acid and glycerol in the AN microplate system
(Biolog
Inc.) enable their separation: P. asaccharolytica is positive for
glyoxylic acid and P.
uenonis
is positive for glycerol (56). P.
asaccharolytica is also positive for α-fucosidase, while
the other
two species are negative. In addition, the differences in the degrees of
pigmentation
may be helpful, with P. uenonis being the most and P. endodontalis the
least
pigmenting
(56). Positive indole, N-acetyl-β-glucosaminidase, and trypsin
reactions comprise
a
typical pattern for P. gingivalis (89).
Unlike most Porphyromonas species, P. bennonis, P.
somerae,
and P. catoniae are indole negative, and the two latter
species also weakly
saccharolytic
(101, 195,196), which may lead to their misidentification as Prevotella species
in
clinical laboratories. However, the sensitivity to vancomycin and
satellite-like growth
pattern
(i.e., larger colonies surrounded by smaller colonies), together with negative
indole
and
α-fucosidase but positive N-acetyl-β-glucosaminidase reactions, identify
the isolate as P.
somerae
(195). Also, P. catoniae isolates grow as satellite-like
colonies but are
nonpigmented,
resistant to vancomycin but susceptible to sodium polyanethol sulfonate in
the
special-potency antimicrobial disk test, and produce major amounts of propionic
acid as
shown
by GLC (101). P. bennonis is a slow-growing organism with very slight
pigmentation
after
extended incubation of at least 10 days and has a variable susceptibility to
vancomycin
(196).
Unlike other Porphyromonas species, it produces major amounts of acetic
and succinic
acids
in GLC, and some strains are catalase positive (196).
Most Porphyromonas species of
animal
origin have been differentiated from the human strains by a positive catalase
reaction
(89). Table
5presents the key reactions for distinguishing Porphyromonas species.
A
small number of Prevotella species produce indole: P. intermedia, P
nigrescens, P. pallens,
P.
micans, and “P. massiliensis” (12, 45, 89).
Except for P. massiliensis, these species are
pigment
producers, as are P. corporis, P. denticola, P. loescheii, P.
melaninogenica, P. shahii,
P.
tannerae, and P. histicola (44, 89).
Some strains of P. histicola have a bull’seye
appearance
due to pigmentation of the colony center (44). Noteworthy
is that pigment
production,
as well as its degree, on BA may take up to 14 days and vary from tan to brown
to
black depending on the species. Detection of lipase on egg yolk agar may be
useful: P.
intermedia
and P. nigrescensand many P. loescheii strains are
positive. Notably, the
separation
of P. intermedia from P. nigrescens is not possible by phenotypic
methods.
Besides
the production of pigment and indole, Prevotella organisms are
characterized by
their
capability of fermenting a variety of sugars. P. massiliensis is an
exception, being very
unreactive;
however, the description of the species is based on one strain
(12). Prevotella
organisms produce acetic and succinic acids as their major metabolic end
products
of glucose fermentation in GLC; however, P. marshii, unusually, also
produces
propionic
acid. Due to the high and still increasing number of Prevotellaspecies,
which have
been
described based on 16S rRNA gene sequencing, their precise identification by
biochemical
tests alone is challenging. Table 6 lists enzyme and
sugar fermentation reactions
that could be of value in
their identification.
Fusobacteria
can be presumptively identified by a limited number of simple laboratory tests,
including
cell morphology with Gram stain, growth in 20% bile, and indole production, but
their
definitive identification to the species level may require additional
biochemical testing
(Table
7). F. nucleatum, appearing as white, speckled, or bread
crumb-like colonies, is
known
for its wide heterogeneity. It includes five subspecies, F. nucleatumsubsp.
animalis, F.
nucleatum
subsp. fusiforme, F. nucleatum subsp. nucleatum, F.
nucleatum
subsp.polymorphum, and F. nucleatum subsp. vincentii,
but phenotypic tests are
not
able to identify the organism to the subspecies level. Also, F.
periodonticum is
indistinguishable
from F. nucleatum by phenotypic methods. In the case of the increasingly
clinically
important F. necrophorum, there are two subspecies, F.
necrophorumsubsp.
funduliforme, isolated from human infections, and F.
necrophorum
subsp. necrophorum from animal infections (20, 87, 160).
Both subspecies are
indole
and lipase positive, but colony morphology and few phenotypic tests, including
erythrocyte
agglutination, are able to distinguish them (87). A
Fusobacterium egg yolk agar
test
has been described for selective isolation of fusobacteria and rapidly
differentiating F.
necrophorum
(127). An indole-negative pleomorphic organism that grows on BBE may be
presumptively
identified as F. mortiferum (89). F.
ulcerans closely resembles indolenegative
F.
varium strains but reduces nitrate. All fusobacteria produce major
amounts of
butyric
acid as their metabolic end product (Table 2)
and, in addition, F. naviforme, F.
russii, and F.
varium produce lactic acid, as detected in GLC (89).
Within
the newly described phylum Synergistetes (92), Jonquetella
anthropi (91)
and Pyramidobacter
piscolens(46) are the two human-derived species that have been
cultured
so far. They are asaccharolytic rods with acetic acid as their major metabolic
end
product
of glucose fermentation. J. anthropi is susceptible to bile and forms
pinpoint colonies
on
blood agar, while the colonies of P. piscolens are somewhat bigger and
highly convex to
pyramidal.
Both species are unreactive to most biochemical tests; however, two reactions
in
the
Rapid ID 32 A system (bioMerieux) are able to separate these species, as J.
anthropi is
positive
for glycine arylamidase and leucyl glycine arylamidase and P. piscolens is
highly
positive
for glycine arylamidase (46, 91).
Dialister
species are coccobacilli that form tiny colonies on blood agar.
They are
asaccharolytic
and grow poorly in liquid media; lack of reactivity in conventional biochemical
tests
hampers their identification. They are often confused with Veillonella because
of their
tiny
cell size. There are a few enzymes in the Rapid ID 32 A kit (bioMerieux) that
may be
useful
in distinguishing three Dialister species: D. pneumosintes is
positive for glycine
arylamidase,
D. micraerophilus for alanine, phenylalanine, serine, and tyrosine
arylamidases
and
arginine dihydrolase, and D. succinatiphilus for alkaline phosphatase (90, 129).
However,
D. invisus and D. propionifaciens are negative for all tests in
this kit. The latter
species
produces propionate, which can be detected by GLC (90).
Molecular methods, such
as
16S rRNA gene sequencing, are often needed for the accurate detection
of Dialister
species in clinical specimens (90, 165).
B.
wadsworthensis isolates are asaccharolytic, bile resistant, and strongly positive
for
catalase,
and most strains are urease positive (11).
This species also produces hydrogen
sulfide,
and its growth is stimulated by bile (ox gall) and pyruvate. This pattern,
together
with
its typical colony characteristics on BBE, may result in its reliable
identification.
Two
human Sutterella species, S. wadsworthensis and S. parvirubra,
and the
novel
Parasutterella excrementihominis are asaccharolytic and very unreactive
organisms, of
which
only S. wadsworthensis has been isolated from infections so far. P.
excrementihominis
and S. parvirubra are strictly anaerobic, have coccoid
cells, and are
negative
for nitrate reduction (135, 173), while S. wadsworthensis is a straight rod that is
able
to grow under microaerobic conditions and to reduce nitrate (211).
Of
the motile gram-negative anaerobic genera isolated from human
specimens,
Selenomonas andAnaerobiospirillum are saccharolytic,
while
Phocaeicola and Desulfovibrio are asaccharolytic. Unlike the
others,Desulfovibrio
species are positive for desulfoviridin, which can be detected by adding
2 N
NaOH to cell paste on a swab and observing for red fluorescence under UV light
(64, 209). Desulfomicrobium
orale is a straight rod and is negative for desulfoviridin but,
typically,
both D. orale and Desulfovibrio species reduce sulfate (106). P.
abscessus is a
slow-growing
coccobacillus with a lophotrichous flagellar arrangement (4).
Flagellae
ofSelenomonas
are arranged on the concave side of the cell, while those of Centipeda are
around
the cell (126).Anaerobiospirillum species have a corkscrew shape and a
jerky
motility.
A. succiniciproducens is sensitive to colistin, and it ferments glucose,
maltose,
lactose,
and sucrose. A. thomasii can be differentiated from A.
succiniciproducens by
carbohydrate
fermentations and by α-glucosidase and β-galactosidase activities, the former
being
negative and the latter positive for both reactions (121). Desulfovibrio
species are
curved
rods with a rapid, progressive motility (except for the nonmotile D. piger)
and are
resistant
to colistin and to 20% bile (except for the bile-sensitive D. desulfuricans)
(209). A
rather
simple test scheme, including catalase, indole, nitrate, and urease, is able to
separate
the
four Desulfovibrio species isolated from human clinical specimens: D.
desulfuricans is
positive
for nitrate and urease, D. fairfieldensis is positive for catalase and
nitrate, and D.
vulgaris
is positive for indole, while D. piger is negative for all
these reactions (209). Table
9presents
an identification scheme for motile gram-negative anaerobic genera.
Advanced Techniques for Identification
Whole-cell bacterial identification by
matrix-assisted laser desorption ionization–time-offlight
mass spectrometry has proven to be a promising
method for the identification of gramnegative
anaerobic bacteria (137, 192).
Peptides and small proteins, which are assumed to
be characteristic for each bacterial species, can
be measured from whole cells, cell lysates,
or crude bacterial extracts. The method is
cost-effective, accurate, and rapid (176), making
it a potential alternative for traditional
identification. However, accurate identification
depends on the unknown organism being present in
the database. There are numerous
unnamed species among the anaerobic gram-negative
bacilli which can confound this type of
analytical approach.
Another method which can reliably detect
as-yet-unnamed taxa as well as identify known
species is 16S rRNA gene sequence analysis (33).
For clinical purposes, a 500-bp sequence
from the 5 end of the gene is able to identify
most, but not all, members of this group to
species level. This method is being increasingly
used for identification of anaerobic bacteria,
because sequencing of the gene is faster and more
accurate than biochemical testing and,
notably, independent of the growth characteristics
(181, 190). Recently, sequencing of
the rpoB gene has also proven its potential
for bacterial identification (1). For
instance, rpoB gene analysis has been
successfully used for distinguishing two closely
related Fusobacterium species, F.
nucleatum and F. periodonticum, and for oral isolates
versus those isolated from intestinal biopsies (193).
However, sequencing, as a routine
method, may not be feasible for many clinical
laboratories. The development of algorithms to
screen for those isolates that can be adequately
identified by conventional methods and to
refer difficult-to-identify isolates for 16S rRNA
gene sequencing has been proposed
(33, 181). Also, commercial 16S rRNA gene sequence-based
identification kits, containing
reagents for DNA extraction and amplification, are
available (MicroSeq; Applied Biosystems,
Foster City, CA). However, they require
instruments,including a thermal cycler and
automated gene sequencer, and software for
interpretation,and the data needed to assess
their value in identifying gram-negative anaerobes
are not available.
Although sequencing of the 16S rRNA gene is a
useful method for identification of fastidious
anaerobic organisms, providing a much faster
turnaround time than conventional methods,
molecular analysis alone should not replace
culturing in the clinical setting. Phenotypic
characteristics, obtained by culture and
biochemical testing, assist in correlating the
sequence-based data, which can be sometimes
difficult to interpret, for example, due to
incomplete sequences stored in the database. More
importantly, culture is necessary for
antibiotic susceptibility testing of isolates from
clinical specimens.
Unculturable Anaerobic Gram-Negative Rods
In many chronic infections, if not in all, several
as-yetuncultivated phylotypes representing
gram-negative anaerobic phyla can be detected.
These include two deep branches of the
phylum Bacteroidetes found in the human
mouth and gut (104, 134) and a similar group
within the newly described phylum Synergistetes
(92), for which no cultivable
representatives are available (206).
In the female genital tract, several Prevotella-like
phylotypes have been strongly associated with
bacterial vaginosis (143). Currently, chronic
venous leg ulcers are considered polymicrobial
infections in which unknown bacteroidales are
among the most ubiquitous organisms (213).
SEROLOGIC TESTS Back to top
Because infections caused by gram-negative
anaerobic taxa are polymicrobial and
opportunistic in nature, serological procedures
are not practical for their identification from
colonies. Furthermore, no standardized tests that
would be useful for these bacteria are
available for the detection of antibodies or
antigens.
ANTIMICROBIAL SUSCEPTIBILITIES Back to top
Trends of increasing resistance among
gram-negative anaerobes to antimicrobial agents
have been observed (77). Although susceptibility
to antibiotics can vary considerably among
species within the same genus, most clinical
laboratories neither perform the accurate
species-level identification of the isolated
organism nor test the susceptibilities of anaerobic
isolates (66). Without knowledge of
the local susceptibility patterns, the choice of proper
antimicrobial therapy can be hampered and make the
treatment outcomes of anaerobic
infections less predictable. According to recent
surveys conducted in the United States,
Europe, Kuwait, Taiwan, and New Zealand, members
of the B. fragilis group are among the
most resistant anaerobes to various antimicrobial
agents; this situation is independent of the
geographical location (61,
86, 115, 144, 162,185, 214). Some variation, however, exists in
resistance rates between countries and areas. In a
large U.S. survey, including 10
geographically diverse medical centers, yearly
changes in the B. fragilis group susceptibility
patterns to ertapenem, imipenem, meropenem,
ampicillin-sulbactam, piperacillintazobactam,
cefoxitin, clindamycin, moxifloxacin, tigecycline,
chloramphenicol, and
metronidazole were followed from 1997 to 2004 (185).
Among the >5,000 isolates tested,
the most resistant organism proved to be a Parabacteroidesspecies,
P. distasonis. Its
resistance rate (17%) to ampicillin-sulbactam
nearly doubled during the follow-up period,
compared to an average resistance rate of 2% for
all the other species combined, during the
later years. Moreover, one of three of the tested P.
distasonis strains proved to be resistant
to cefoxitin. Of the B. fragilis group
organisms, B. fragilis was the most susceptible (<2% of
the tested strains resistant) to carbapenems and
β-lactam–β-lactamase inhibitor
combinations (185). In another large
survey, conducted at the National Taiwan University
Hospital, the proportion of susceptible isolates
of Bacteroides,
Prevotella, and/orFusobacterium species to many antimicrobials,
especially cefmetazole,
clindamycin, and the combination
ampicillin-sulbactam, decreased during the period from
2000 to 2007 (115). Noteworthy was the
presence of intermediate or resistant strains to
carbapenems among the tested B. fragilis,
Fusobacterium, and Prevotellaisolates from blood;
one B. fragilis isolate was even resistant
to all four carbapenems tested (115). Indeed, blood
isolates seem to be less susceptible than those
from intra-abdominal, obstetric, or other
infections (3). Newer fluoroquinolones
have been considered to have good antianaerobic
effects, but this situation may be worsening among
gram-negative anaerobes
(63, 145). Pseudoflavonifractor capillosus (formerly
a non-fragilis group Bacteroides) has
shown to be prevalent and to exhibit high
resistance rates to moxifloxacin in Greece (145),
with the caveat that the isolates examined in the
study were not identified using sequencebased
methods. In general, carbapenems, some
β-lactam–β-lactamase inhibitor
combinations, chloramphenicol, and metronidazole
are the most useful antianaerobic agents,
whereas most cephalosporins, clindamycin, and most
fluoroquinolones are currently
considered less active for use against
gram-negative anaerobic bacteria in severe infections
(3, 61, 77, 86, 115, 144, 185, 212). In addition, the relatively new antimicrobial
drugs
tigecycline and linezolid have demonstrated good
antianaerobic effects against gramnegative
anaerobes; however, some resistant Bacteroides and
Prevotella strains have been
reported (65, 185,
214). A few cases of multidrug-resistant B.
fragilis isolated from blood
and drainage fluid after gastric surgery have been
reported (94, 208), with the strains being
resistant to antianaerobic agents, such as
carbapenems, β-lactam–β-lactamase inhibitors,
clindamycin, and/or metronidazole. Interestingly,
one of these cases was successfully treated
with linezolid (208). Despite the extensive
use of metronidazole against anaerobes, acquired
resistance has been considered rare (<5%).
However, prolonged exposure of nim genecarrying
Bacteroidesspecies to metronidazole can select for therapeutic resistance (59).
A
considerable number of Dialister strains,
isolated from a variety of clinical specimens,
showed decreased susceptibility to metronidazole
but without harboring nim genes (128).
Also, rare strains of Prevotella species
may appear highly resistant to metronidazole,
resulting in poor treatment outcome (131).
Although the agar dilution method is recommended
as the method of choice for susceptibility
testing of anaerobic species, many studies have
shown that the Etest (bioMerieux) provides
reliable results on susceptibilities of anaerobic
isolates from clinical specimens
(59, 61, 86, 214). The Etest is simple to perform and readily
available when needed, offering
a useful method for susceptibility testing in
clinical microbiology laboratories. It has been
observed that some slow-growing
metronidazole-resistant clones can be overlooked when
using the standard incubation time of 48 h;
therefore, it has been recommended that
laboratories reexamine the Etest plates after 72 h
to look for small colonies within the
metronidazole inhibition zone (59,131).
Susceptibility testing methods for anaerobic bacteria
are described further in chapter 72.
Table 10 summarizes the current antimicrobial a ctivity rates of the
clinically most relevant
gram-negative anaerobic taxa.
EVALUATION, INTERPRETATION, AND REPORTING OF
RESULTS Back to top
Knowledge about the resident microbiota and
awareness of the role of anaerobic bacteria in
disease permit the clinician to anticipate the
likely infecting species at different body sites.
Training for anaerobic techniques, in general, and
introduction of more advanced methods
for the detection and precise identification of
anaerobic organisms are urgently needed in
clinical microbiology laboratories, considering
the current reports of increasing frequencies of
anaerobic bacteremia and septicemia (108),
increasing numbers of patients withF.
necrophorum-associated invasive diseases (20), and also the increasing
resistance rates of
anaerobes to various antimicrobials (77,
86, 115, 185, 212, 214). In cases of inaccurate
microbiology and inappropriate choices of
antimicrobial agents in treating an infection,
mortality rates and other treatment failures
increase significantly (142, 174). Collecting of
clinical specimens should avoid the mucosal microbiota,
and proper transport medium and
times should be used for keeping the anaerobes
alive. Factors such as foul-smelling
discharge, proximity of infection to mucosal
surfaces, abscess formation, and necrotic tissue
indicate the presence of anaerobes in the
specimen. A definitive identification of an anaerobic
isolate should be obtained for all isolates from
normally sterile body sites, including blood,
spinal fluid, and organs or body cavities; when
the patient is gravely ill and not responding to
treatment; and when prolonged treatment is
necessary. It would be desirable for reference
laboratories to periodically provide information
on local susceptibility patterns of anaerobic
species within the clinically important taxa.
However, not only an accurate antimicrobial
therapy but also proper surgery, such as the
drainage of abscesses and excision of necrotic
tissue, are important in resolving anaerobic
infections. It is notable that chronic infectious
lesions, in particular,include consortia of
bacteria organized in biofilms.
The most important, and often difficult, task in
the reporting of results of the isolation of
gram-negative anaerobes is determination of
whether the organism is involved in the
infectious process or is merely a bystander
originating from the patient’s commensal
microbiota. All isolations from normally sterile
sites should be regarded as significant. A
secondary challenge is that these organisms are
often found in polymicrobial infections, with
a number of different species present and often as
a biofilm. Obtaining pure cultures for
susceptibility testing can therefore be
time-consuming, and the resulting susceptibility
profiles may be conflicting, making the
recommendation of appropriate antimicrobial therapy
difficult. Fortunately, such infections typically
respond well to empirical treatment, as partial
disruption of the bacterial consortium responsible
is sufficient to allow the body’s defenses to
deal with the infection.
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