TAXONOMY Back to top
The bacteria discussed in this chapter all belong
to the class Actinobacteria, the genera of
which are characterized by specific 16S rRNA gene
signature nucleotides (144, 168)
belonging to the lineage of gram-positive bacteria
with high guanine-plus-cytosine (G+C)
contents, with the exception of the genusExiguobacterium,
which belongs to the
class Firmicutes, the members of which have
low G+C contents. The coryneform bacteria are
most diverse and are differentiated by
chemotaxonomic features (Table 1). Phylogenetic
investigations, in particular, 16S rRNA gene
sequencing, have in general confirmed the
framework set by chemotaxonomic investigations.
The 16S rRNA gene sequencing data
demonstrate that the genera Corynebacterium and
Turicella are more closely related to the
partially acid-fast bacteria and to the genus Mycobacterium
than to the other coryneform
organisms covered in this chapter (71,
108, 133). The genus Arthrobacter, which contains
rods, is phylogenetically intermixed with the
genus Micrococcus (and genera formerly
called Micrococcus), which contains cocci
(see chapter 19) (54, 84).
The
genus Rothia contains both rod-forming
organisms, represented by Rothia dentocariosa, and
a coccus-forming species, Rothia mucilaginosa, formerly
Stomatococcus mucilaginosus (24).
Other genera which are phylogenetically closely
related include Oerskovia,
Cellulosimicrobium, and Cellulomonas (14,
64, 137, 143), as well
as Arcanobacteriumand Actinomyces (109).
DESCRIPTIONS OF THE GENERA Back to top
Genus Corynebacterium
The number of species belonging to the genus Corynebacterium
has dramatically increased
from 22 in 1990 to 81 species (and one taxon
group) at the time of writing, 50 of which (and
one taxon group) are medically relevant. Clinically
relevant Corynebacterium species validly
described since the last edition of this Manualinclude
C. canis (50), C. freiburgense (53),
C.
hansenii (119), C. massiliense (96),
C. pilbarense (3), C. pyruviciproducens (151),
C.
sputi (164), C. stationis (9),
C. timonense (96), and C. ureicelerivorans (163).
The cell walls of corynebacteria contain meso-
diaminopimelic acid (m-DAP) as the diamino
acid as well as short-chain mycolic acids with 22
to 36 carbon atoms (22). The medically
relevant Corynebacterium species C.
amycolatum, C. atypicum, and C. kroppenstedtii as well
as C. caspium (from seals) and C.
ciconiae (from storks) lack mycolates (21, 23,
77). The
corynebacterial cell wall contains arabinose and
galactose (22). Palmitic (C16:0), oleic
(C18:1ω9c), and stearic (C18:0) acids are the main
cellular fatty acids (CFAs) in all
corynebacteria, and tuberculostearic acid (TBSA)
can be detected in some medically
relevant Corynebacterium species (e.g., C.
urealyticum, C. kroppenstedtii, C. confusum, C.
appendicis, and C. minutissimum) (7, 23,
61, 165). The G+C contents
of Corynebacterium spp. vary from 46 to 74
mol%, indicating the enormous diversity within
this genus. The phylogenetic relationships within
the genus Corynebacterium have been
outlined previously (108,133),
creating an extensive and reliable database for future
comparative 16S rRNA gene studies, e.g., for the
delineation of new species. Complete
genome sequences for Corynebacterium
diphtheriae (17),Corynebacterium
jeikeium (148), C. urealyticum (150),
and C. kroppenstedtii (149) have been described
previously.
Gram staining of corynebacteria shows slightly
curved, gram-positive rods with sides not
parallel and sometimes slightly wider ends, giving
some of the bacteria a typical club shape
(Fig.
1a). Corynebacteria whose
morphologies differ from this morphology include C. canis,
C. durum, C. matruchotii, and C. sundsvallense(see below under each
species’ name). Cells
generally stain evenly. If Corynebacterium cells
are taken from fluid media, they are
arranged as single cells, in pairs, in V forms, in
palisades, or in clusters with a so-called
Chinese-letter appearance. It is again emphasized
that the club-shaped form of the rods is
observed only for true Corynebacterium spp.
Genus Turicella
The genus Turicella is phylogenetically
closely related to the genus Corynebacterium but
contains Turicella otitidis as the only
species. The cell wall contains m-DAP, but mycolic acids
are not present (71). The main CFAs for T.
otitidis are the same as those
for Corynebacterium spp., but all T.
otitidis strains also contain significant amounts of TBSA
(2 to 10% of all CFAs) (71).
T. otitidis is the only coryneform bacterium that has a polar lipid
profile without glycolipids. The G+C content varies
between 65 and 72 mol% (71). Gram
staining shows relatively long gram-positive rods
(Fig. 1b). T. otitidis is catalase positive,
nonmotile, and an oxidizer.
Genus Arthrobacter
The genera Arthrobacter and Micrococcus are
so closely related phylogenetically that it has
been stated that micrococci are, in fact,
arthrobacters which are unable to express rod forms
(84). Presently, the genusArthrobacter contains
over 50 species, of which only a few have
been recovered from human clinical specimens (92).
Lysine is the diamino acid of the cell
wall, and C15:0ai is the overall dominating CFA;
it represents more than 50% of all CFAs in
most Arthrobacter species. The G+C contents
vary between 59 and 70 mol%, indicating the
diversity within this genus.
Gram staining may demonstrate a rod-coccus cycle
(i.e., rod forms in younger cultures and
cocci in older colonies) when cells are grown on
rich media (e.g., Columbia base agar).
Jointed rods (i.e., rods in a rectangular form
that contributed to the designation of this genus
as “arthros,” which means “joint” in ancient
Greek) may also be observed in younger
cultures (i.e., at 24 h) but may not be
demonstrable for every species. Arthrobacters are
catalase positive, their motility is variable, and
they are always oxidizers.
Genus Brevibacterium
The genus Brevibacterium presently
comprises 22 species, of which 9 species are medically
relevant. m-DAP is the diamino acid type
found in the cell wall. C15:0ai and C17:0ai usually
represent more than 75% of all CFAs (48).
The G+C contents vary between 60 and 70
mol%.
Gram staining demonstrates relatively short rods,
which may develop into cocci when
cultures become older (after 3 days).
Brevibacteria are catalase positive, nonmotile, and
oxidizers.
Genus Dermabacter
The genus Dermabacter presently comprises
only one species, D. hominis. m-DAP is the
diamino acid of the cell wall, and C15:0ai and
C17:0ai usually account for 40 to 60% of all CFAs.
The G+C content range is between 60 and 62 mol% (81).
Gram staining shows very short
rods (Fig. 1c), which are often
initially misinterpreted as cocci. D. hominis strains are
catalase positive, nonmotile, and glucose
fermenters.
Genus Helcobacillus
The genus Helcobacillus, part of the family
Dermabacteraceae, has been described for an
isolate recovered from a patient with a cutaneous
discharge presenting with an erythrasma.
Interestingly, phenotypic features of the
bacterium were initially thought to be suggestive
of C. minutissimum but later, by a
polyphasic approach, of D. hominis. The Gram stain
shows gram-positive, straight, short (0.7 to 1.0
μm in length by 0.4 to 0.7 μm in diameter),
irregular rods. Colonies are apigmented. The type
strain is catalase positive, nonmotile, and
a glucose fermenter. This taxon is amycolated,
with a G+C content of 68.6 mol%; m-DAP is
the cell wall diamino acid, with the CFAs C15:0ai
, C17:0ai , and C16:0i (together, ~70% of the
total) predominating (122).
Genus Rothia
The genus Rothia is also included in this
chapter because some species are rod-like. The
genus Rothia belongs to the family Micrococcaceae.
Collins and colleagues
reclassified Stomatococcus mucilaginosus as
Rothia mucilaginosa (24). Since Rothia
mucilaginosa exhibits coccoid forms in Gram stains, the genus Rothia is
also covered
in chapter 19 (on the catalase-positive
gram-positive cocci). However, the species Rothia
dentocariosaclearly exhibits mainly rod forms and is, therefore, covered in
this chapter on
coryneform bacteria.
Lysine is the diamino acid of the cell wall, and
C15:0ai and C17:0ai usually represent 40 to 60%
of all CFAs. The G+C contents range between 47 and
56 mol%. Rothia strains can be quite
pleomorphic by Gram staining, but filamentous
forms are normally not observed. They have
a variable catalase reaction, are nonmotile, and
exhibit a fermentative metabolism.
Genus Exiguobacterium
The genus Exiguobacterium is
phylogenetically related to the so-called “group 2 bacilli” (39).
Presently, 13 species are included in this genus,
of which only E. acetylicum and E.
aurantiacum have been mentioned in publications as being isolated from human
clinical
material. Lysine is the diamino acid of the cell
wall, and C15:0ai and C17:0ai comprise only about
30 to 40% of the total CFAs. E. acetylicum contains
significant amounts of C13:0 and C13:0ai,
which are not found in any other coryneform taxon
(7). The G+C content is about 47 mol%.
Exiguobacteria present as relatively short rods in
young cultures. Strains are catalase
positive and motile and have a fermentative
metabolism.
Genus Oerskovia
In older textbooks, oerskoviae were assigned to
the nocardioform group of organisms due to
their morphological features. This includes
branching vegetative substrate hyphae and
penetration into agar but no aerial hyphae.
However, there is now phylogenetic and
chemotaxonomic evidence that Oerskovia, including
the reclassified
genus Promicromonospora (143),
is more closely related to genera like Cellulomonas than to
the mycolic acid-containing genera like Nocardia.
Representatives of the type
species, Oerskovia turbata, were recovered
from soil, but human pathogens originally
identified as O. turbata have now been
placed inCellulosimicrobium funkei (14). Lysine is the
diamino acid of the cell wall, and C15:0ai is the
main CFA in oerskoviae. The G+C content is 70
to 75 mol%.
Gram staining shows coccoid-to-rod-shaped bacteria
which originate from the breaking up of
mycelia.Oerskovia strains are catalase
positive, their motility is variable, and they are
fermentative.
Genus Cellulomonas
The genus Cellulomonas presently comprises
17 species, of which only C. hominis and C.
denverensis have been described as being isolated from humans (13,
64, 105). Ornithine is
the diamino acid of the cell wall, and C15:0ai and
C16:0 are the main CFAs. The G+C content is
71 to 76 mol%.
Gram staining shows small, thin rods. All Cellulomonas
spp., except C. fermentans and C.
humilata, are catalase positive, their motility is variable, the
environmental Cellulomonas strains are
cellulolytic (whereas C. hominisdid not hydrolyze
cellulose in the test system used) (64),
and they have a fermentative metabolism.
Genus Cellulosimicrobium
The Cellulosimicrobium genus presently
comprises three species. The medically relevant
species C. cellulanshad been designated Cellulomonas
cellulans or Oerskovia
xanthineolytica in the past (137). The reason for removing C. cellulans from
the
genus Cellulomonas was that the topology of
the 16S rRNA gene dendrogram indicated that
the branching point of this taxon was outside Cellulomonas
proper. In addition, the
chemotaxonomic characteristic of lysine as diamino
acid supported reclassification.
Predominant CFAs include C15:0ai, C15:0i, C16:0i,
and C16:0. The major menaquinone (MK) is MK-
9(H4), and the G+C content is 74 mol%. It should
be noted that the
genus Cellulosimicrobium is related to the
genus Oerskovia but is nevertheless distinct.
In young cultures, a mycelium that fragments later
into irregular, curved, and club-shaped
rods is produced. Catalase activity is detected,
and strains are nonmotile. All strains have a
fermentative metabolism.
Genus Microbacterium
It has been known since the mid-1990s that the
genera Microbacterium and Aureobacterium
are phylogenetically intermixed (114), and the
diamino acid in the third position of the
tetrapeptide of the peptidoglycan was considered a
most important chemotaxonomical marker. L-Lysine
is present in microbacteria and Dornithine
in the former aureobacteria, a phenomenon noted
for some genera
(e.g.,Propionibacterium and Bifidobacterium),
where there is not a good correlation between
the type of the diamino acid in the peptidoglycan
layer and their phylogenetic positioning.
Because a set of signature nucleotides within the
16S rRNA genes of both microbacteria and
aureobacteria could be demonstrated, it was
proposed that both genera be unified in a
redefined genus, Microbacterium (145).
Now, over 50 Microbacterium species have
been validly named, but only a minority of them
has been demonstrated to be of clinical importance
(74). Microbacteria are most frequently
encountered in environmental specimens (e.g.,
soil). C15:0ai and C17:0ai are the two main
CFAs, often representing up to 75% of the total
CFAs (51, 73, 145). The G+C content
of Microbacterium spp. is 65 to 76 mol%,
indicating the diversity within the genus.
Gram staining often shows thin or short rods with
no branching. Catalase activity and
motility are variable. Microbacteria can be either
fermenters or oxidizers.
Genus Curtobacterium
Curtobacterium spp., like microbacteria, belong to the peptidoglycan type B
actinomycetes
(i.e., cross-linkage exists between positions 2
and 4 of the two peptide subunits). Ornithine
is the diamino acid and the only amino acid
composing the interpeptide bridge. Curtobacteria
have an acetyl peptidoglycan acyl type and MK-9 as
the major MK, whereas microbacteria
possess a glycolyl type and MK-11,12 (Table 1). For most curtobacteria, C15:0ai and
C17:0ai represent more than 75% of all CFAs (46).
The G+C contents range from 68 to 75
mol%. Presently, nine Curtobacterium species
are validly described.
Gram staining shows small and short rods with no
branching. Catalase activity is positive,
motility is observed in most strains, and all
strains show a respiratory metabolism which
proceeds slowly in oxidizing carbohydrates.
Genus Leifsonia
The former “Corynebacterium aquaticum” was
transferred into the
genus Leifsonia as Leifsonia aquatica in
2000 (38) and is the only medically relevant species
in this genus. L. aquatica strains belong
to the peptidoglycan B-type actinomycetes and,
therefore, cannot be true corynebacteria, which
actually possess an A-type peptidoglycan
(i.e., cross-linkage exists between positions 3
and 4 of the two peptide subunits).
Diaminobutyric acid is the diamino acid of the
cell wall peptidoglycan, and C15:0ai and
C17:0ai are the main CFAs, as with microbacteria,
but represent <75% of all CFAs (73). The
G+C content is about 70 mol%.
Gram staining shows thin rods. The strains are
catalase and oxidase positive (the latter
feature is atypical for coryneform bacteria),
always motile, and oxidizers.
Other Unusual Coryneforms
Recent examination of coryneform bacteria using
primarily sequence-based identification
approaches have shown that additional genera can
be recovered from human clinical
materials. These are briefly mentioned below.
Genus Janibacter
Strains of the genus Janibacter (94),
the first medically relevant coryneform reported from
the familyIntrasporangiaceae, were found to
be associated with bacteremia (36, 91; K. A.
Bernard, unpublished observation). Janibacter strains
can have Gram stain-variable or grampositive
coccoidal-to-rod-like forms in singles, pairs, or
irregular clumps. Their DNA base
composition is 69 to 73 mol% G+C, with an unusual
CFA profile consisting of significant
volumes of the CFAs C16:0i, C17:1, and C17:0.
These bacteria are described as oxidizers, with
white, creamy, or yellowish pigments. They are
nonmotile, and optimal growth may occur at
25 to 30°C.
Genus Pseudoclavibacter
The Pseudoclavibacter genus was first
described in 2004 to accommodate the misidentified
type strain of “ Brevibacterium helvolum” (93).
Shortly afterwards, the novel
genus Zimmermannella was proposed but was
found to be an illegitimate, later, homotypic
synonym of Pseudoclavibacter(http://www.bacterio.cict.fr/xz/zimmermannella.html).
Pseudoclavibacter alba strains were recovered from urine, and Pseudoclavibacter
bifida strains were recovered from blood cultures and wounds. By Gram
staining, these
species were short or medium-length gram-positive
rods, with P. bifida demonstrating some
rudimentary branching. Their DNA base composition
is 62 to 68 mol% G+C. Major CFAs are
C15:0ai, C16:0, C16:0i, and C17:0ai. These strains
are oxidizers, with white or yellowish colonies
(93). Optimal growth occurs at 30°C.
Genera Brachybacterium and Knoellia
Isolates from the genus Brachybacterium and
one strain from the genus Knoellia, all
recovered from blood cultures, have been
characterized (K. A. Bernard, unpublished
observations). There are currently 12 species in
the genus Brachybacterium, which is part of
the family Dermabacter aceae, and so they
are most closely related to the
genus Dermabacter. Members of this genus
grow at 37°C, exhibit gram-positive coccoidal
and rod-like forms, and have a G+C content of 68
to 73 mol%. The Brachybacterium blood
culture isolates are metabolically fermentative
and have branched-chain-type CFAs. The
genus Knoellia, like that of the genus Janibacter,
is a member of the
family Intrasporangiaceae. Cells are
irregular gram-positive rods or cocci with major CFAs of
the branched-chain type, and their G+C content is
68 to 69 mol%. The single Knoellia blood
culture isolate is capnophilic, growing best in 5%
CO2 at 37°C.
Genus Arcanobacterium
The Arcanobacterium genus presently
contains nine species, of which A. haemolyticum, A.
bernardiae, and A. pyogenes have been recovered from human clinical
specimens. Lysine is
the diamino acid of the cell wall, whereas in the
phylogenetically closely
related Actinomyces spp., lysine or
ornithine is found. Arcanobacteria contain MKs of the MK-
9(H4) type, whereas the Actinomyces spp.
examined so far have MK-10(H4). The main CFAs
of arcanobacteria are C16:0, C18:1ω9c, and C18:0
(as in Corynebacterium spp. and T. otitidis),
but unlike with corynebacteria, significant
amounts of C10:0, C12:0, and C14:0 may also be
detected (7). The G+C content is 48
to 52 mol%.
Gram staining of arcanobacteria shows irregular,
gram-positive rods. All clinically relevant
arcanobacteria are catalase negative; they are
nonmotile and fermenters.
Genus Gardnerella
The genus Gardnerella does not have a
particular phylogenetic relationship to any of the
established genera described in this chapter. It
is remotely related to the
genus Bifidobacterium (97),
and these genera share some important features, such as
production of acetic and lactic acids as
fermentation products. G. vaginalis is the only species
belonging to the genus Gardnerella. Studies
of the ultrastructure of the cell wall of G.
vaginalishave demonstrated that it has a cell wall similar to but much
thinner than the cell
walls of other gram-positive bacteria (i.e., there
is a smaller peptidoglycan layer) (134).
Lysine is the diamino acid of the cell wall, and
CFAs are similar to those detected
in Actinomyces spp., Arcanobacterium spp.,
and Corynebacterium spp., with C16:0and
C18:1ω9c predominating. The G+C content of 42 to
44 mol% is lower than that of every other
genus described in this chapter.
Gram stains show thin Gram stain-variable rods or
coccobacilli (Fig. 1d). Catalase is not
produced, and cells are nonmotile and have a slow
fermentative metabolism.
EPIDEMIOLOGY AND TRANSMISSION Back to top
Many species of the corynebacteria are part of the
normal biota of the skin and mucous
membranes in humans and other mammals. The habitat
for some medically irrelevant
corynebacteria (e.g., C. terpenotabidumand C.
halotolerans) is the environment. It is
noteworthy that not all corynebacteria are equally
distributed over skin and mucous
membranes; many of them occupy a specific niche. C.
diphtheriae can be isolated from the
nasopharynx as well as from skin lesions, which
actually represent a reservoir for the spread
of diphtheria. Important opportunistic pathogens
like C. amycolatum, Corynebacterium
striatum, and D. hominisare part of the normal human skin biota but
have thus far not been
recovered from throat swabs from healthy
individuals (158). Coryneform bacteria prominent
in the oropharynx include Corynebacterium durum
and R.
dentocariosa (158). Corynebacterium auris and T. otitidis
seem to have an almost exclusive
preference for the external auditory canal. In
nearly every instance that Corynebacterium
macginleyi has been isolated, eye specimens have been the source (62).
Another Corynebacterium species with a
distinctive niche isCorynebacterium
glucuronolyticum, which is almost exclusively isolated from
genitourinary specimens from
humans (47) and from animals (28).
C. urealyticum, another genitourinary pathogen, has,
like C. jeikeium,been cultured from
inanimate hospital environments.
The natural habitat of arcanobacteria is not fully
understood, but A. haemolyticum is
recovered from throat as well as from wound swabs,
whereas A. bernardiae has been found
mainly in abscesses adjacent to skin (G. Funke and
K. Bernard, unpublished observations). It
is unclear whether the two species are part of the
normal skin and/or the gastrointestinal
biota. A. pyogenes is found on mucous
membranes of cattle, sheep, and swine. Brevibacteria
can be found on dairy products (e.g., cheese) but
are also inhabitants of the human skin
(48). Arthrobacters are some of the most frequently
isolated bacteria when soil samples are
cultured, but at least Arthrobacter cumminsii also
seems to be present on human skin
(63, 92). Members of the generaExiguobacterium,
Oerskovia, Cellulomonas,
Cellulosimicrobium, and Microbacterium have their habitats in
the inanimate environment
(e.g., soil and activated sludge). Microbacterium
spp. have also been recovered from hospital
environments (51). Curtobacteria are
primarily plant pathogens (46).
G. vaginalis can be found in the anorectal biota of healthy adults of both
sexes as well as in
that of children (16). It is also part of the
endogenous vaginal biota in women of
reproductive age. The optimal pH for the growth of
G. vaginalis is between 6 and 7, i.e., at
elevated pH in the vagina. The organism can also
be recovered from the urethras of the male
partners of women with bacterial vaginosis (BV) (16).
CLINICAL SIGNIFICANCE Back to top
Estimating the clinical significance of coryneform
bacteria isolated from clinical specimens is
often confusing for clinical microbiologists. This
is in part due to the natural habitat of
coryneform bacteria, which may lead to their
recovery if specimens were not taken correctly.
The reader is referred to the guidelines on
minimal microbiological requirements in
publications on disease associations of coryneform
bacteria (72).
Coryneform bacteria should be identified to the
species level if they are isolated (i) from
normally sterile body sites, e.g., blood (but not
if only one of multiple specimens becomes
positive), (ii) from adequately collected clinical
material if they are the predominant
organisms, and (iii) from urine specimens if they
are the only bacteria encountered and the
bacterial count is >104/ml or if they are the
predominant organisms and the total bacterial
count is >105/ml.
The clinical significance of coryneform bacteria
is strengthened by the following findings: (i)
multiple specimens are positive for the same
coryneform bacteria; (ii) coryneform bacteria
are seen in direct Gram stains, and a strong
leukocyte reaction is also observed; and (iii)
other organisms recovered from the same material
are of low pathogenicity.
For a comprehensive summary of case reports on
individual coryneform bacteria, the reader
is referred to review articles (6, 72).
Historically,
diphtheria caused by C. diphtheriae (or Corynebacterium ulcerans)
has been the
most
prominent infectious disease for which coryneform bacteria are responsible.
Therefore,
special
attention is given to that disease in this chapter. Due to immunization
programs, the
disease
has nearly disappeared in countries with high socioeconomic standards. However,
the
disease is still endemic in some subtropical and tropical countries as well as
among
individuals
of certain ethnic groups (e.g., indigenous peoples in the Americas and
Australia).
In
the 1990s, diphtheria reemerged in the states of the former Soviet Union.
However,
despite
increased global travel activities, only a few imported cases have been
reported by
countries
with well-developed health care systems.
The
main manifestation of diphtheria is an upper respiratory tract illness
accompanied by a
sore
throat, dysphagia, lymphadenitis, low-grade fever, malaise, and headache. A
nasopharynx-adherent
membrane which may occasionally lead to obstruction is
characteristic.
The severe systemic effects of diphtheria include myocarditis, neuritis, and
kidney
damage caused by the C. diphtheriae exotoxin, which is encoded by a
bacteriophage
carrying
the tox gene. C. diphtheriae may also cause cutaneous diphtheria
or endocarditis
(with
either toxin-positive or toxin-negative strains). Some people with poor
hygienic
standards
(e.g., drug and alcohol abusers) are prone to colonization (on the skin more
often
than
in the pharynx) by C. diphtheriaestrains, which are often nontoxigenic.
G.
vaginalis is associated with BV. Its causative role in the syndrome is
controversial
(141, 142),
as it is certainly not the sole cause; other bacteria, like Atopobium
vaginae,
Leptotrichia/Sneathia
species, andMegasphaera-like bacteria are also involved in
BV
(44, 155).
Recurrent BV is due to reinfection rather than to relapse (i.e., overgrowth of
the
previously
colonizing biotype). In pregnant women, BV may lead to preterm birth, premature
rupture
of membranes, and chorioamnionitis (16). G.
vaginalis may also be recovered from
cultures
of blood from patients with postpartum or postabortal fevers and may also cause
infections
in newborns. Although it might be recovered from the urethras of males, its
disease
association in males is usually questionable. Cases involving serious
infections
(septicemia,
wound infections) in sites other than those associated with the genital tract
or
obstetrics
are rare but have been reported, including in men (87).
COLLECTION, TRANSPORT, AND STORAGE OF
SPECIMENS Back to top
In
general, coryneform bacteria do not need special handling when samples are
collected.
The
general principles outlined in chapters 9 and 16 in
this Manual apply to this group of
organisms
as well.
C.
diphtheriae
The
diagnosis of diphtheria is primarily a clinical one. The physician should
notify the
receiving
laboratory immediately of suspected diphtheria. In case of respiratory
diphtheria,
material
for culture should be obtained on a swab (either a cotton- or a
polyester-tipped
swab)
from the inflamed areas in the nasopharynx. Multisite sampling (nasopharynx) is
thought
to increase sensitivity. If membranes are present and can be removed (swabs
from
beneath
the membrane are most valuable), they should also be sent to the microbiology
laboratory
(although C. diphtheriae might not be culturable from those in every instance).
Nasopharyngeal
swabs should be obtained from suspected carriers. It is preferable that the
swabs
are immediately transferred to the microbiology laboratory for culturing. If
the swabs
must
be sent to the laboratory, semisolid transport media (e.g., Amies) ensure the
maintenance
of the bacteria. All coryneform bacteria are relatively resistant to drying and
moderate
temperature changes. Material from patients with suspected cases of wound
diphtheria
can be obtained by swab or aspiration.
Vaginal
and extravaginal specimens for culturing G. vaginalis can be collected
with cottontipped
swabs.
It is best to take one swab for direct examination and to take another swab for
culture
if necessary, such as for epidemiologic studies. If culture media cannot be
directly
inoculated,
then the swab should be placed in a transport medium (e.g., Amies) and culture
should
be done within 24 h. It is noteworthy that G. vaginalis is susceptible
to sodium
polyanethol
sulfonate (SPS), so an SPS-free medium (or an SPS medium supplemented with
gelatin)
should be used in order to achieve optimal recovery of G. vaginalis from
blood
culture
systems whenever G. vaginalis is suspected.
Long-term
preservation in skim milk at −70°C is applicable to all coryneform bacteria.
Except
with samples containing lipophilic corynebacteria, the same skim milk tube can
be
repeatedly
thawed and frozen (G. Funke, unpublished observation). For nonlipophilic
coryneforms,
good results were also observed with Microbank tubes (Pro Lab Diagnostics,
Austin,
TX) (G. Funke, unpublished observation). The advantage of using these tubes is
that
individual
beads can be taken out of the tube. Coryneform bacteria can also be stored for
decades
when they are kept lyophilized in an appropriate medium (e.g., 0.9% NaCl
containing
2% bovine serum albumin).
DIRECT EXAMINATION Back
to top
After
the appropriate isolation media have been inoculated (see below under
“Isolation
Procedures”),
the swabs taken from diphtheritic membranes may be subjected to Neisser or
Loeffler
methylene blue staining. A positive stain is characterized by metachromatic
granules
(polar
bodies). However, it is noteworthy that the sensitivity of the microscopic
examination
is
limited. Antigen assays for the direct detection of coryneform bacteria are not
recommended.
As
described previously, C. diphtheriae, C. ulcerans, and C.
pseudotuberculosis are the only
species
able to harbor the bacteriophage which carries the diphtheria tox gene
and
potentially
produce diphtheria toxin (72). PCR-based,
direct-detection systems for the
diphtheria
tox gene have been described, using methods to detect fragment A and/or
the
entire
tox gene (32, 107) or fragment A and B subunits of the tox gene (101).
The system
described
by Nakao and Popovic had the highest sensitivity when Dacron polyester-tipped
swabs
were used and when silica gel packages were stored at 4°C rather than at room
temperature
(101). Conventional PCR detection of the regulatory dtxR gene
has been
evaluated
(111). Detection of the tox gene using a real-time platform has
been outlined
(15, 99, 136).
Real-time detection using primers targeting the C. diphtheriae tox gene
for C.
ulcerans
strains have required modifications (15, 135, 136).
Direct detection of the
diphtheria
tox or dtxR gene as the sole test of clinical specimens has not
been
recommended,
as expression of diphtheria toxin must be demonstrated (some strains may
harbor
but not express the gene), and so microbiological culture is essential for
confirming
diphtheria
(33).
The
“gold standard” for the diagnosis of BV is direct examination of vaginal
secretions and
not
the culture ofG. vaginalis, since G. vaginalis can also be
recovered from healthy women.
A
bedside test for BV is examination of the vaginal discharge to detect the typical
“fishy”
trimethylamine
odor, which is enhanced after alkalinization with 10% KOH (but G.
vaginalis
is not responsible for the amine production). The typical smear of
vaginal discharge
from
BV patients shows “clue cells” (bacteria covering epithelial cell margins)
together with a
mixed
biota consisting of large numbers of small gram-negative
(predominantly
Prevotella and Porphyromonasspp.) and gram-variable (G.
vaginalis) rods
and
coccobacilli, whereas lactobacilli are almost always absent. It is recommended
that a
standardized
Gram staining interpretative scheme be used in order to improve the
reproducibility
of this method (104, 142). Although not recommended for routine laboratory
procedures,
the isolation of G. vaginalis can support the diagnosis of BV.
ISOLATION PROCEDURES Back
to top
Coryneform
bacteria, including C. diphtheriae, can be readily isolated from a 5%
sheep blood
agar
(SBA)-based selective medium containing 100 μg of fosfomycin per ml (plus 12.5
μg of
glucose-6-phosphate
per ml), since nearly all coryneforms (except Actinomyces spp. and D.
hominis)
are highly resistant to this compound (152).
It is also possible to put disks
containing
50 μg of fosfomycin (plus 50 μg of glucose-6-phosphate [already incorporated in
the
disk]) (BD Diagnostics, Sparks, MD) on an SBA plate and then examine the
colonies
which
grow around the disk. Selective media for coryneform bacteria containing 50 to
100
μg/ml
furazolidone (Sigma, St. Louis, MO) have also been described. If lipophilic
corynebacteria
like C. jeikeium or C. urealyticum are sought, then 0.1 to 1.0%
Tween 80
(Merck,
Darmstadt, Germany) could be added to an SBA plate (add Tween 80 before pouring
the
medium). Additional methods to demonstrate lipophilia are described below.
Medically
relevant
coryneforms described to date do not grow on MacConkey agar. However, if
“coryneform”
bacteria are recovered from this medium, they should be examined carefully to
rule
out rapidly growing mycobacteria.
With
very few exceptions (some arthrobacters, microbacteria, and curtobacteria,
which have
optimal
growth temperatures of between 30 and 35°C), the medically relevant coryneform
bacteria
grow at 37°C. It is desirable to culture specimens for coryneform bacteria in a
CO2-
enriched
atmosphere since some taxa, e.g.,Rothia and Arcanobacterium spp.,
grow much
better
under those conditions. Nearly all medically relevant coryneform bacteria grow
within
48
h, so that primary culture plates should not be incubated longer than that.
However, if
liquid
media are used (e.g., for specimens from normally sterile body sites),
specimens
should
be held for 5 days before the culture is declared negative. Final Gram staining
and
subculture
should be performed only with turbid broths.
It
is recommended that urine specimens be incubated for longer than 24 h to check
for the
presence
of C. urealyticum but only when patients are symptomatic or have
alkaline urine or
struvite
crystals in their urine sediment.
C.
diphtheriae
The
primary plating media for the cultivation of C. diphtheriae should be
SBA plus one
selective
medium (e.g., cystine-tellurite blood agar [CTBA] or freshly prepared Tinsdale
medium)
(33, 34). If silica gel is used as a transport medium, the desiccated
swabs need to
be
additionally incubated overnight in broth (supplemented with either plasma or
blood),
which
should then be streaked onto the primary plating medium. The plates are read
after
18
to 24 h of incubation at 37°C, preferably in a 5% CO2-enriched atmosphere.
Tellurite
inhibits
the growth of many noncoryneform bacteria, but even a few C. diphtheriae strains
are
sensitive to potassium tellurite and will therefore not grow on CTBA but may
grow on
SBA.
It is noteworthy that growth on CTBA and tellurite reduction are not specific
for C.
diphtheriae,
since many other coryneforms may also produce black (albeit
smaller) colonies.
The
best medium for direct culturing of C. diphtheriae is Tinsdale medium (34).
However, the
limitations
of Tinsdale medium are its relatively short shelf life (<4 weeks) and the
necessity
to
add horse serum to it. On Tinsdale plates, both tellurite reductase activity
(as shown by
black
colonies) and cystinase activity (as shown by a brown halo around the colonies)
can be
observed.
If neither CTBA nor Tinsdale medium is available, colistin-nalidixic acid blood
agar
plates
are recommended for the isolation of C. diphtheriae or any other
coryneform
bacterium.
It is necessary to pick multiple colonies from colistin-nalidixic acid blood
agar
plates
to rule out C. diphtheriae (first by Gram staining and then by
subculturing, with
subsequent
biochemical testing). Nonselective Loeffler serum slants are no longer
recommended
for the primary isolation of C. diphtheriae because of overgrowth by
other
bacteria.
G.
vaginalis
Vaginal
swabs are cultured on vaginalis agar (see chapter 17 in
this Manual for the
preparation)
and should be semiquantitatively streaked out with a loop. Incubation is at 35
to
37°C in a 5% CO2-enriched atmosphere or in a candle jar. Slight beta-hemolysis
is
observed
on human or rabbit blood-containing media but not on SBA (on which G.
vaginalis
can also grow, exhibiting alpha-hemolysis). Plates may be checked
for the growth
of
diffuse beta-hemolytic colonies of <0.5 mm in diameter after 24 h, but very
often G.
vaginalis
is best observed after 48 h. Gram staining of the suspected
colonies confirms the
diagnosis
of G. vaginalis.
IDENTIFICATION Back to top
Basic
tests available in every microbiology laboratory are of great value for the
identification
of coryneform
bacteria. The Gram staining morphology of the cells can exclude the
assignment
to many genera and may even lead to the assignment to the correct genus (e.g.,
to
the genus Corynebacterium, Turicella, orDermabacter) (Fig.
1). Morphology, size,
pigment,
odor, and hemolysis of colonies are also valuable criteria in the differential
diagnosis
of coryneform bacteria.
von
Graevenitz and Funke (157) outlined a biochemical identification system for coryneform
bacteria
which was based on previous results from the Centers for Disease Control and
Prevention’s
(CDC’s) Special Bacteriology Reference Laboratory (79).
This system includes
testing
for catalase; fermentation or oxidation (which is best observed in semisolid
cystine-
Trypticase
agar medium rather than on triple sugar iron or oxidation-fermentation media,
with
fermentation indicated by acid or alkali production in the entire tube and
oxidation
found
at the surface of the tube); motility; nitrate reduction (24 h of incubation);
urea
hydrolysis
(24 h of incubation); esculin hydrolysis (up to 48 h of incubation); acid
production
from
glucose, maltose, sucrose, mannitol, and xylose (48 h of incubation); CAMP
reaction
(24
h of incubation) with a beta-hemolysin-producing strain of Staphylococcus
aureus (e.g.,
strain
ATCC 25923), i.e., with a positive reaction indicated by an augmentation of the
effect
of S.
aureus beta-hemolysin on erythrocytes, resulting in a complete hemolysis in
an
arrowhead
configuration (Fig. 2); and lipophilia (24 h of incubation), the test for which is
performed
only for catalase-positive colonies of <0.5 mm in diameter. For the test for
lipophilia,
colonies are subcultured onto ordinary SBA and onto a 0.1%- to 1%-Tween 80-
containing
SBA plate. Lipophilic corynebacteria develop colonies up to 2 mm in diameter
after
24 h on Tween 80-supplemented agar. It has also been suggested that levels of
growth
in
brain heart infusion broth with and without supplementation of 1% (vol/vol)
sterile Tween
80
be compared and that strains which grow only in the supplemented broth can be
called
lipophilic.
The identification protocols given in this chapter are, in principle, based on
the
identification system of von
Graevenitz and Funke (157) (Tables 3 and 4).
Manually
performed identification panels include the API (formerly RapID) Coryne system
(bioMerieux,
Marcy l’Etoile, France) and the RapID CB Plus system (Remel, Lenexa, KS),
which
are widely used. The API Coryne system contains 50 taxa in its present database
(version
3.0), and comparison to an online database, APIWEB, is available
(https://apiweb.biomerieux.com).
In a comprehensive multicenter study, it was found that
90.5%
of the strains belonging to the taxa included were correctly identified, with
additional
tests
needed for correct identification for 55.1% of all strains tested (69).
Reproducible
results
are best obtained if the manufacturer’s recommendations for use are rigorously
followed.
It was concluded that the system is a useful tool for the identification of the
diverse
group
of coryneform bacteria encountered in routine clinical laboratories. The RapID
CB Plus
system
correctly identified 80.9% of the strains to the genus and species levels and
an
additional
12.2% to the genus level, but with less accurate species designations; it was
also
concluded
that this system may perform well under the conditions of a routine clinical
laboratory
(67). An updated version of a panel for the automated Vitek system
(bioMerieux)
has
been described (120). However, it is always important to question critically the
identifications
provided by any commercial identification system and to correlate the results
with
simple basic characteristics, such as macroscopic morphology and Gram staining
results.
Furthermore, it is important to note that for both commercially available
manual
identification
systems, the databases have not been updated since the end of the1990s, and
therefore,
the recently described taxa are not covered.
For
some identifications, the commercial API 50CH system (bioMerieux) has been found
to
be
useful. For example, when the AUX medium (usually attached to the kit for
gram-negative
nonfermenters
[bioMerieux]) is applied to the API 50CH system, utilization reactions which
allow
the differentiation of Brevibacterium spp. or some Arthrobacter spp.
can be observed
(48, 54).
A
reference laboratory also uses chromatographic techniques for further
characterization of
coryneform
bacteria. The presence of mycolic acids and their chain lengths can be detected
by
thin-layer chromatography (TLC), gas chromatography, and mass spectrometry or
highperformance
liquid
chromatography (27). These methods can be useful for the differentiation
of Corynebacterium
spp. (mycolic acids of 22 to 36 carbon atoms) from the partially acid-fast
bacteria
(mycolic acids of 30 to 78 carbon atoms) but may also provide evidence that a
coryneform
bacterium is not a Corynebacterium (exceptions include C. amycolatum,
C.
atypicum,
and C. kroppenstedtii) if mycolic acids are not detected.
The detection of the
diamino
acid of the peptidoglycan by one-dimensional TLC is of certain value for
determining
the
genus to which a particular strain belongs (Table 1).
In some cases, partial hydrolysates
of
the peptidoglycan are separated by two-dimensional TLC to reveal the
interpeptide bridge
of
the peptidoglycan in order to distinguish between genera having the same
diamino acid in
the
peptide moiety. For example, some of the yellow-pigmented microbacteria and all
curtobacteria
have ornithine as their diamino acids, but microbacteria have (glycine)-
ornithine
as the interpeptide bridge, whereas curtobacteria possess ornithine only.
The
analysis of CFAs by means of gas-liquid chromatography with the Sherlock system
(MIDI
Inc.,
Newark, DE) is a useful method for the identification of coryneform bacteria.
This
system
is, in general, able to correctly identify coryneform bacteria to the genus
level, but
identification
to the species level is, in most cases, impossible, although the commercial
database
suggests that it is possible. This is due to the very closely similar CFA
profiles
obtained
for coryneform bacteria belonging to the same genus (7)
and because the
quantitative
profiles observed strongly depend on the incubation conditions. When a
laboratory
creates an individual database based on its own entries, species identification
becomes
more likely (K. A. Bernard, unpublished observation). The mycolic acids of some
corynebacteria
(e.g., C. auris) are cleaved at the temperature (300°C) produced in the
injection
port of the system, resulting in peaks being misidentified as fatty acids,
e.g.,
C17:1v6c
to Cv9c, by the Sherlock system (7, 57).
Molecular
genetics-based identification systems for coryneform bacteria have been
outlined
in
recent years. Restriction fragment length polymorphism analysis of the partly
amplified
and
digested 16S rRNA gene has been demonstrated to be of use for the
identification of
species,
e.g., within the genus Corynebacterium (154).
Some corynebacteria may also be
identified
to the species level by examination of the length of the 16S-23S rRNA
intergenic
spacer
region (4). A very useful approach for the identification of true
corynebacteria is the
sequencing
of a 434- to 452-bp fragment of the rpoB gene (using primers designated
C2700F
and C3130R), since this particular region of the gene displays a high degree of
polymorphism
within the genusCorynebacterium (82, 83). A
divergence of >5% within this
particular
part of the rpoB genes of two compared strains suggests that they belong
to two
different
species. For more definitive taxonomic investigations of coryneforms, and in
cases
of
the growth of coryneform bacteria from difficult-to-obtain clinical material (132),
fulllength
16S
rRNA gene sequencing might be indicated. Determination of the complete 16S
rRNA
gene sequence is a rational approach for identifying corynebacteria, since most
established
species exhibit 3% or greater divergence, except for C. afermentans, C.
coyleae,
C.
mucifaciens (<2% divergent); C. aurimucosum, C. minutissimum, and C.
singulare
(<2%); C. sundsvallense and C. thomssenii (<1.5%);
C. ulcerans and C.
pseudotuberculosis
(<1% divergent from each other and both <2% divergent from C.
diphtheriae); C.
propinquum and C. pseudodiphtheriticum (<2%); C. xerosis, C.
freneyi,
and C. hansenii (<2%); and C. macginleyiand C.
accolens (<2%).
In
very few selected cases (i.e., if the divergence of the 16S rRNA genes is
≤1.3%),
quantitative
DNA-DNA hybridizations might be necessary, with sequencing of the
complete
rpoB gene recently being suggested as a substitute for that approach (2).
Because
of
the ever-growing number of coryneform taxa encountered in clinical specimens,
it has
become
difficult to readily differentiate these taxa by biochemical means alone, so
sequencing
studies are likely to replace some of the biochemical testing in the near
future. It
is
emphasized that unidentifiable, clinically significant coryneform bacteria
should be sent to
an
established reference laboratory experienced in corynebacterial identification
for
characterization
which includes sequence-based analyses.
IDENTIFICATION: DESCRIPTIONS OF GENERA AND
SPECIES Back to top
Genus Corynebacterium
C.
accolens
C.
accolens (103) is found in specimens from the eyes, ears, nose, and oropharynx.
Endocarditis
of native aortic and mitral valves due to this agent has been described.
Colonies
are,
as for all other lipophilic corynebacteria, convex, smooth, and <0.5 mm in
diameter on
SBA.
C. accolens strains had initially been described to exhibit satellitism
in the vicinity of S.
aureus
strains, attributable to its lipophilism (for the method
recommended to demonstrate
lipophilism,
see above under “Identification”). C. accolens has a variable
pyrazinamidase
reaction
but is negative for alkaline phosphatase, which differentiates it from the
morphologically
and biochemically closely related species C. tuberculostearicum (Table
3).
The
API Coryne and RapID CB Plus systems correctly identify C. accolens (67, 69). C.
accolens strains
are susceptible to a broad spectrum of antibiotics.
C.
afermentans subsp. afermentans
C.
afermentans subsp. afermentans (125) is
part of the normal human skin biota and has so
far
been isolated mainly from blood cultures. Colonies are whitish, convex with
regular
edges,
creamy, and about 1 to 1.5 mm in diameter after 24 h of incubation. C.
afermentans
subsp. afermentans has an oxidative metabolism. The API
Coryne system
provides
the numerical code 2100004 for this species. About 60% of all strains of this
taxon
are
CAMP reaction positive. C. afermentans subsp. afermentans can be
differentiated from C.
auris
and T. otitidis (both of which give the same API Coryne
numerical code) by the
consistency
of its colonies (C. auris is slightly adherent to agar) and morphology
upon Gram
staining
(T. otitidis has longer cells). By chemo taxonomic means, both C.
afermentans
subspecies and C. auris contain mycolates, whereas T.
otitidis lacks them, but
this
technique is not applicable in routine clinical laboratories. C.
afermentans
subsp. afermentans is generally susceptible to β-lactam
antibiotics, but
resistance
to erythromycin, clarithromycin, azithromycin, and clindamycin have been
reported
(42).
C.
afermentans subsp. lipophilum
Strains
belonging to the species C. afermentans subsp. lipophilum (125)
have been isolated
mainly
from blood cultures but also from superficial wounds. Colonies are, typically
for
lipophilic
corynebacteria, convex, smooth, and <0.5 mm in diameter after 24 h. C.
afermentans
subsp. lipophilum has an oxidative metabolism and does not
produce acid from
any
of the carbohydrates usually tested (Table 3).
It is the only species of nonfermenting,
lipophilic
corynebacteria which may exhibit a positive CAMP reaction. C.
afermentans
subsp.lipophilum is not included in the API Coryne
database. The numerical
profile
observed for the species is 2100004 and so by that method cannot be discerned
from
more
robustly growing C. afermentans subsp. afermentans, C. auris, or T.
otitidis, which
have
the same code as well. Strains are usually susceptible to β-lactam antibiotics.
C.
amycolatum
C.
amycolatum is part of the normal human skin biota but was not recovered from
throat
swabs
from healthy persons (158). C. amycolatum is the most frequently
encountered
Corynebacterium species in human clinical material (56).
It is also the most
frequently
isolated nonlipophilic Corynebacterium in dairy cows with mastitis (80). C.
amycolatum
strains are nearly always multidrug resistant (68).
Colonies are very typically
dry,
waxy, and grayish white with irregular edges and are 1 to 2 mm in diameter
after 24 h
of
incubation (Fig. 3a). C. amycolatum actually has a fermentative metabolism,
but when
cystine-Trypticase
agar media are used for the observation of acid production from
carbohydrates,
C. amycolatum appears to resemble an oxidizer (i.e., to have main acid
production
at the surface of the medium). Strains of C. amycolatum are remarkable
for their
variability
in basic biochemical reactions (Table 3)
and are often misidentified as the
biochemically
similar species C. xerosis, C. striatum, or C. minutissimum (56, 161, 170).
These
species can be differentiated by the following means. C. amycolatum and C.
minutissimum
do not grow at 20°C, but C. xerosis and C. striatum do;
in addition, C.
xerosis
does not ferment glucose at 42°C, whereas the other three species
do; and C.
minutissimum
and C. striatum produce alkali from formate but C.
amycolatum and C.
xerosis
do not (161). When tested on Mueller-Hinton agar supplemented with 5% sheep
blood,
nearly all C. amycolatum strains were resistant to the vibriocidal
compound O/129
(150-μg
disks) (Oxoid, Basingstoke, United Kingdom), as indicated by there being no
zone of
inhibition
around the disk (56). In contrast, only 4% of all C. amycolatumstrains were
resistant
to O/129 when tested on Mueller-Hinton agar with 5% horse blood (80).
The API
Coryne
system identifies this species well, but in every case additional reactions
must be
carried
out in order to confirm the identification of C. amycolatum (69).
All C.
amycolatum
strains produce propionic acid as the major end product of glucose
metabolism.
In
contrast to many other corynebacteria, C. amycolatum exhibits only weak
or no leucine
arylamidase
activity. The identification may also be suggested by the absence of mycolic
acids.
In addition, it may be shown that acyl phosphatidylglycerol is a major
phospholipid
in C.
amycolatum, unlike in other Corynebacterium spp., in which other
phospholipids are
predominant (153).
C.
appendicis
The
one strain of C. appendicis described in the literature was isolated
from a patient with
appendicitis
accompanied with abscess formation (165).
This lipophilic species contains large
amounts
of TBSA (up to 50% of all CFAs), which is not seen in any
other
Corynebacterium species. It is differentiated from CDC coryneform group
F-1 bacteria
by a
positive alkaline phosphatase reaction but negative reactions for nitrate
reduction and
sucrose
fermentation.
C.
argentoratense
C.
argentoratense (127) has been isolated from the human throat and was once recovered
from
a blood culture (8). Colonies are cream colored, nonhemolytic, slightly rough, and 2
mm
in diameter after 48 h of incubation. Phenotypically, C. argentoratense may
appear to be
very
similar to (rare) ribose-negative strains ofC. coyleae. However, glucose
fermentation
by C.
argentoratense is quite rapid, compared to that of the slowly fermenting
species C.
coyleae.
In addition, CAMP reaction-negative C. argentoratense produces
propionic acid as a
fermentation
product, but CAMP reaction-positive C. coyleae does not (8). C.
argentoratense
is the only medically relevant Corynebacterium species
expressing α-
chymotrypsin
activity, which can be observed in the API ZYM (bioMerieux) system; however,
the
blood culture isolate was not observed to produce that enzyme (8).
Although C.
argentoratense
is phylogenetically closely related to C. diphtheriae, it
does not harbor
the toxgene,
coding for the diphtheria toxin.
C.
atypicum
Although
C. atypicum clearly belongs to the genus Corynebacterium,
corynomycolic acids, as
in C.
amycolatumand C. kroppenstedtii, are not detected (77). C.
atypicum is not lipophilic
but
shows only pinpoint-sized colonies after 48 h of incubation. It is the only
medically
relevant
Corynebacterium not expressing pyrazinamidase but expressing β-
glucuronidase
activity.
C.
aurimucosum
The
initial description of C. aurimucosum was based on a single strain which
exhibited
slightly
yellow and sticky colonies on 5% SBA plates but had colorless and slimy colonies
on
Trypticase
soy agar without blood (166). Biochemically, this particular C. aurimucosum strain
was
similar to C. minutissimum. The number of C. aurimucosum strains
was significantly
enhanced
when it was demonstrated that some former CDC coryneform group 4 bacteria
actually
belong to C. aurimucosum (26).
It is important to note that strains of C.
aurimucosumexhibit
a grayish-black pigment which is not seen in any other
true
Corynebacterium. Strains originally designated “Corynebacterium
nigricans” were later
shown
to be C. aurimucosum (26). Some strains of R.
dentocariosa can also exhibit a
charcoal-black
pigment (26); these strains are differentiated from C. aurimucosum by
being
constantly
nitrate reductase positive, having a possibly negative catalase reaction, and
having
branched-chained CFAs, as opposed to straight-chain-type CFAs in C.
aurimucosum.
API
Coryne codes for pigmented C. aurimucosum strains include 0000125,
2000125, and
2100327.
Phylogenetically, C. aurimucosum is closely related (>98.8% identity)
to both C.
singulare
and C. minutissimum by 16S rRNA gene sequencing but can
readily be discerned
by
partial rpoB gene sequence analysis.
C.
auris
C.
auris (57) has almost exclusively been isolated from the ear region.
Colonies are dryish
and slightly
adherent to agar but do not penetrate agar; they become slightly yellowish with
time,
and they have diameters ranging from 1 to 2 mm after 48 h of incubation. C.
auris
does not produce acid from any carbohydrates usually tested. All C.
auris strains are
strongly
CAMP test positive. The API Coryne system provides the numerical code 2100004
for
this species. Abundant degradation products of mycolic acids are indirectly
observed
when
CFA patterns are determined with the Sherlock system (57).
It is noteworthy that the
MICs
of β-lactam antibiotics for C. auris strains are elevated, but the
molecular mechanism
for
this is not known at present (68).
C.
bovis
Occasionally,
human infections have been attributed to the lipophilic bovine species C.
bovis.
Characterization
of lipophilic corynebacteria solely on the basis of the results of phenotypic
tests,
in the absence of modern polyphasic methods or identification schemes, was
probably
incorrect
(Table 3). This species had not been definitively recovered for many years
from
human
clinical material (72). Recently, an oxidase-positive, human blood culture isolate of C.
bovis
was identified based on a polyphasic approach, with an API Coryne
code of 0101104
(8),
as was an isolate from a prosthetic joint infection (1).
C.
canis
This
nonlipophilic Corynebacterium was isolated from a wound infection after
a dog bite (50).
It
has some unusual microscopic features in that it exhibits very filamentous rods
(>15 μm
in
length), and some cells even show branching. C. canis is esculinase and
α-glucosidase
positive
and is the only Corynebacterium expressing trypsin activity.
C.
confusum
C.
confusum has been isolated from patients with foot infections, a blood
culture (61), and a
breast
abscess (8). Colonies are whitish, glistening, convex, creamy, and up to 1.5
mm in
diameter
after 48 h. Acid from glucose is produced only very weakly, becoming visible in
the
API
Coryne or the API 50CH gallery only after 48 to 72 h. Weak growth under
anaerobic
conditions
corresponds to slow fermentative acid production. It is advisable to incubate
the
API
Coryne system after 24 h for another day in those cases in which the results
for acid
production
are ambiguous (i.e., with only a slight change in the color of the indicator).
After
48 h
of incubation, the API Coryne system provides the numerical code 3100304 for
this
species;
the breast abscess strain had a code of 3100104. Interestingly, the breast
abscess
strain
was also CAMP reaction positive, making it potentially more difficult to discern
from C.
coyleae
isolates (8). C. confusum is correctly identified by the RapID CB Plus
system (67). If
glucose
fermentation is judged to be negative, C. confusum strains can be
misidentified as C.
propinquum.
However, in contrast to that species, C. confusum does not
hydrolyze tyrosine
and
contains small amounts of TBSA (1 to 3%), whereas C. propinquum hydrolyzes
tyrosine
but
does not contain TBSA. C. confusum is differentiated from C. coyleae and
C.
argentoratense
by its ability to reduce nitrate.
C.
coyleae
C.
coyleae (65) has been isolated mainly from cultures of blood and other
normally sterile
body
fluids, but it may also be recovered from a variety of sterile body fluids,
abscesses, and
urogenital
specimens (8, 40). Colonies are whitish and slightly glistening, with entire
edges,
and
are about 1 mm in diameter after 24 h. The consistency of the colonies is
either creamy
or
sticky. A slow fermentative acid production from glucose and a strongly
positive CAMP
reaction
are the most significant phenotypic characteristics. C. coyleae is
positive for cystine
arylamidase,
which is not observed for many other corynebacteria. Various API Coryne
numerical
codes have been observed, especially 2100304 and 6100304. C. coyleae is
always
positive
for ribose fermentation, whereas the biochemically similar species C.
argentoratense
varies in this reaction. The API Coryne database lists only 6% of
glucosefermenting
C.
coyleae strains, and therefore, when applying this commercial
identification
system,
the clinical microbiologist may not receive a correct identification (69).
However, the
two
numerical profiles given above combined with a positive CAMP reaction are
highly
indicative
of C. coyleae. This species is correctly identified by the RapID CB Plus
system (67).
Macrolide
resistance has been reported (42).
CDC Group F-1 Bacteria
The
lipophilic CDC group F-1 bacteria (128)
have not been given a species name. Although
genetically
distinct, no distinguishing phenotypic markers which clearly allow their
separation
from
other defined Corynebacteriumspp. have been found. The characteristics
of the CDC
group
F-1 bacteria are consistent with the definition of the genus Corynebacterium
in all
respects.
Of note is the negative alkaline phosphatase reaction (Table
3). CDC group F-1
strains
are usually susceptible to penicillin but are often resistant to macrolides.
C.
diphtheriae
In
2003, the complete genome sequence of a Corynebacterium diphtheriae strain
representative
of the diphtheria outbreak in the former Soviet Union states in the 1990s was
determined
(17). The genome consists of a single circular chromosome of
2,488,635 bp with
no
plasmids. A complete set of enzymes for the glycolysis, gluconeogenesis, and
pentosephosphate
pathways
is present, as are all the de novo amino acid biosynthesis pathways.
Fimbrial
and fimbria-related genes, sialidase (neuraminidase) genes, and iron uptake
systems
have been detected as pathogenicity factors.
C.
diphtheriae is commonly divided into four biotypes, gravis, mitis, belfanti,
and
intermedius;
biotype differentiation is recommended by the WHO (33, 34),
although biotypes
cannot
be assigned separate subspecies status, and biotyping is not satisfactory for
epidemiologic
tracking. Initially, these biotypes were defined by differences in colony
morphology
and biochemical reactions (Table 3). However, only C.
diphtheriaebiotype
intermedius
can be identified on the basis of colonial morphology (small, gray, or
translucent
lipophilic
colonies) as well as positivity for dextrin fermentation. Other C.
diphtheriae
biotypes produce larger (up to 2 mm after 24 h) white or opaque
colonies (Fig.
3b)
which are indistinguishable from each other. The lipophilic C. diphtheriae biotype
intermedius
occurs only rarely in clinical infections, and C. diphtheriae biotype
belfanti
strains
rarely express diphtheria toxin.
Presumptive
identification of C. diphtheriae (as well as of C. pseudotuberculosis
and C.
ulcerans)
may be made by testing suspicious gram-positive rods for the presence of
cystinase
(as detected by using freshly prepared Tinsdale medium or diagnostic tablets
[Rosco,
Taastrup, Denmark]) and the absence of pyrazinamidase (diagnostic tablets are
available
from Key Scientific Products, Stamford, TX). The API Coryne system identifiesC.
diphtheriae
strains, but additional tests are needed for the differentiation
of C.
diphtheriae
biotype mitis, C. diphtheriae biotype belfanti, and C.
diphtheriae biotype
intermedius
(69). Usually, C. diphtheriae strains do not ferment sucrose,
but in Brazil,
sucrose-positive
strains have been described. Large amounts of propionic acid are produced
as
the end product of glucose metabolism. C. diphtheriae strains are
distinct from all other
coryneform
bacteria (except C. pseudotuberculosis and C. ulcerans) in their
CFA patterns by
the
presence of a large volume of C16:1v7c (7).
Diphtheria Toxin Testing
It
is recommended that at least 10 colonies of C. diphtheriae and related
species be tested
for diphtheria
toxin by the Elek method, modified as described by Engler et al. (37),
in a
laboratory
with skill in performing the test and in interpreting the test results. The
modified
Elek
method as described by the WHO Diphtheria Reference Unit was initially used to
characterize
strains from the epidemic in the 1990s in Russia and Ukraine and was found to
be
faster and less technically problematic than the original version. Antitoxin
from various
suppliers
(Berna Biotech AG [Switzerland], Mikrogen [Russia], Biomed [Russia], Pasteur
Merieux/Aventis
Pasteur [France], BulBio-NCIPD [Bulgaria], Instituto Butantan [Brazil], or
Refik
Saydam National Hygiene Centre [Turkey]) (102)
applied to blank filter disks at 10
IU/disk
have been successfully used with the modified Elek test.
PCR-based
methods for the detection of the diphtheria toxin gene (tox) in isolated
bacteria
have
been developed and validated (32, 78, 107).
Conventional PCR detection of the
regulatory
dtxR gene has been evaluated (111).
Detection of the tox gene using a real-time
platform
has been described (15, 99, 136). Primers targeting the C. diphtheriae tox gene
for C.
ulcerans strains perform better with modifications (15, 135,136). tox
PCR assays
applied
directly to clinical specimens are acceptable, particularly because isolation
is not
always
possible for patients already receiving antibiotics. However, a PCR-positive
patient
from
whom bacteria are not isolated or who lacks a histopathologic diagnosis and an
epidemiologic
linkage to a patient with a laboratory-confirmed case of diphtheria should be
classified
as a “probable case” of diphtheria, since to date there are insufficient data
to
conclude
that a PCR-positive result always infers diphtheria. Also, detection of the
toxin gene
in
samples by PCR cannot automatically be attributed to one species because C.
diphtheriae
as well as C. ulcerans and C. pseudotuberculosis may
harbor the bacteriophage
which
carries the diphtheria toxin gene. Furthermore, tox-containing,
nontoxigenic isolates
have
been described and characterized further. Difficulties in identifying C.
diphtheriae and in
correctly
performing toxigenicity tests have recently been demonstrated by an external
quality
control program observing 23 national diphtheria reference centers in Europe;
in
these
centers, 21% of specimens were misidentified and 13% of toxigenicity reports
were
determined
to be unacceptable (102).
Nontoxigenic
strains of C. diphtheriae, i.e., those which do not express toxin in the
Elek test
or
those which lack a detectable diphtheria toxin gene by PCR, have also caused
serious
disease,
such as cases or outbreaks of skin disease, endocarditis, and occasional
mortality
among
homeless people, alcoholics, and intravenous drug abusers (45, 116, 131).
For
nontoxigenic
C. diphtheriae strains circulating in the United Kingdom, it has been
shown that
the
diphtheria toxin repressor (dtxR) genes are functional, so that if these
strains are
lysogenized
by a bacteriophage, they could represent a reservoir for toxigenic C.
diphtheriae
(29).
Antibiotic
treatment, with antibiotics of choice being penicillins or macrolides, is
required to
eliminate
C. diphtheriae and prevent its spread; however, it is not a substitute
for antitoxin
prevention.
Sporadic isolates ofC. diphtheriae resistant to erythromycin or rifampin
have
been
reported.
C.
durum
C.
durum (126) was originally described as being exclusively isolated from
respiratory tract
specimens.
Well- characterized isolates have now been recovered from additional sites,
including
the gingiva, blood cultures, and abscesses (115). C.
durum strains were originally
isolated
after 2 to 3 days from nonselective buffered charcoal-yeast extract plates
inoculated
with
sputa or bronchial washings, but isolates grow well on other standard
laboratory
media.
C. durum is the most frequent Corynebacterium isolated from
throat swabs of healthy
persons
(158). Its pathogenic potential remains unclear. C. durum is a
peculiar nonlipophilic
organism
that forms colonies of only 0.5 to 1 mm in diameter after aerobic incubation
for 72
h.
The original description of this bacterium cited beige and rough colonies with
convolutions,
an
irregular margin, and strong adherence to agar if grown under aerobic
conditions (126).
However,
strains were later described to be sometimes smoother and not necessarily
adherent
to agar (115). Gram staining of aerobic cultures shows long and filamentous
rods,
with
occasional “bulges,” but true C. durum isolates do not have C.
matruchotii-like “whip
handles”
(Fig. 1e and f). Long forms are not otherwise found among
other
Corynebacterium species (except C. canis), nor are they observed
for C. durum when
cells
are grown in a 10% CO2-enriched atmosphere (126).
Strains grow only weakly under
anaerobic
conditions. They always reduce nitrate, and some may exhibit weak and delayed
urease
and esculinase activities. The majority (but not all) of C. durum strains
ferment
mannitol,
which is another very unusual feature for true corynebacteria (Table
3). API
Coryne
codes observed for C. durum include 3000135, 3001135, 3040135, 3400115,
3400135,
3400305, 3400325, and 3400335 (126), as well as 3040325,
3040335, 3440335,
and
3441335 (115). This suggests that most strains are negative for alkaline
phosphatase,
and
all appear to be negative for pyrrolidonyl arylamidase. Only a small number of C.
durum
strains have been tested with the RapID CB Plus system, and all
were correctly
identified
(67). It is most likely that some strains identified as C.
matruchotii in the past may
actually
have been C. durum strains and that differentiation can be difficult if
phenotypic
methods
alone are used. Both species produce propionic acid as a fermentation product
(6). C.
durum usually ferments galactose and very often mannitol, whereas C.
matruchotii is
usually
negative for those sugars. The C. matruchotii type strain exhibits
α-glucosidase
activity,
which is not observed in C. durum (126).
It has been shown that some C.
durum
strains also express β-galactosidase activity and ferment ribose (158).
C. falsenii
C.
falsenii strains (139) have been isolated mainly from sterile body fluids. Colonies are
whitish,
glistening, and smooth, with entire edges, and are 1 to 2 mm in diameter after
24 h.
After
72 h, most strains described to date exhibit a yellowish pigment which becomes
even
more
intense after 120 h. The most characteristic biochemical features of C.
falsenii are a
slow
but fermentative acid production from glucose, a weak pyrazinamidase reaction,
and a
weak
urease activity which becomes visible in either Christensen’s urea broth or the
API
Coryne
system after overnight incubation only. API Coryne codes observed for C.
falsenii
have been 2101104 and 2101304 (8, 139).
C.
freiburgense
This
nonlipophilic species has probably been transmitted to a human by a dog bite (53).
The
5-day-old
colonies exhibit a very peculiar “spoked-wheel” macroscopic morphology not
observed
in other true corynebacteria but observed in some R. dentocariosa strains.
Colonies
are
also strongly adherent to blood agar. Distinct biochemical features are the
lack of
pyrazinamidase
activity and a positive β- galactosidase reaction.
C.
freneyi
This
species was initially outlined on the basis of a study of three strains (117)
which came
from
skin-related material. There is now evidence that C. freneyi is also
isolated from
genitourinary
specimens (52) and bacteremic patients (5). C.
freneyi is phylogenetically
closely
related to C. xerosis. Colonies are typically wrinkled, whitish, dry,
and rough, have
irregular
edges, and are 0.5 to 1 mm in diameter after 48 h incubation, but C. freneyi
strains
are
nonlipophilic. The basic biochemical profile (Table 3) is
similar to that of C. xerosis. AllC.
freneyi
strains studied so far exhibit α-glucosidase activity, which is
not frequently observed
in
otherCorynebacterium species (very few C. amycolatum and all C.
xerosis strains express
this
enzyme). C. freneyi can be furthermore differentiated from C. xerosis
by glucose
fermentation
at 42°C and growth at 20°C, whereas C. xerosis is negative for these two
reactions.
This species is also closely related to C. hansenii.
C.
glucuronolyticum
C.
seminale is a later synonym of C. glucuronolyticum (28, 47).
This species is probably part
of
the normal genitourinary biota of males, while its presence in females is
uncertain.
Recoveries
from blood cultures have been documented (8).
Colonies are whitish yellow,
convex,
and creamy, and they are 1 to 1.5 mm in diameter after 24 h. The fermentative
species
C. glucuronolyticum is remarkable for its variability in basic
biochemical reactions
(Table
3). It is the only medically relevant, large-colony Corynebacterium
species exhibiting
β-glucuronidase
activity. When urease activity is present, it is abundant in Christensen’s urea
broth,
becoming positive after only 5 min of incubation at room temperature (47). C.
glucuronolyticum
is also one of the very few corynebacteria which are able to
hydrolyze
esculin.
All C. glucuronolyticum strains are CAMP reaction positive (Fig.
2). The API Coryne
strip
identifies C. glucuronolyticum well but not strains which are alkaline
phosphatase
positive
(69), although profiles obtained from human strains may differ from
those of animal
isolates
(28). Propionic acid is one of the major end products of glucose
metabolism. C.
glucuronolyticumstrains
are often tetracycline resistant and may also exhibit resistance to
macrolides
and lincosamides (68). 16S rRNA gene sequences derived from fluids of patients
with
prostatitis have been found to be homologous with sequences derived for this
species,
indicating
that C. glucuronolyticum might be involved in selected cases of
prostatitis (146).
C.
hansenii
This
nonlipophilic species exhibits yellow and dry colonies (119).
Biochemical identification
reactions
are similar to those of C. freneyi and C. xerosis. However, C.
hansenii can be
distinguished
from these species by being negative for alkaline phosphatase and α-
glucosidase
reactions. These three species are not well discerned by either 16S rRNA or
partial
rpoB gene sequencing but were found to represent different taxa by
DNA-DNA
hybridization
(119).
C.
imitans
C.
imitans was originally isolated from a nasopharyngeal specimen of a child
suspected of
having
throat diphtheria, as well as from three adult contacts (49).
This was the first welldocumented
case
of the person-to-person transmission of a Corynebacterium species other
than
C. diphtheriae in a nonhospital setting. Additional strains of C.
imitans have been
recovered
from blood cultures (8). Colonies are creamy, whitish gray, and glistening, with
entire
edges, and they are 1 to 2 mm in diameter. The strain does not produce a brown
halo
on
Tinsdale medium but is tellurite reductase positive. Interestingly, it is
positive for polar
bodies
by Neisser staining. Pyrazinamidase activity is only weak, as is fermentation
of
sucrose,
which may lead to an initial misidentification as an atypical C. diphtheriae
strain. It
is
possible that C. imitans may have been misidentified as C.
minutissimum in the past, since
the
biochemical reactions of both taxa are similar (Table 3).
However, C. imitans is CAMP
reaction
positive and does not hydrolyze tyrosine, whereas the opposite reactions are
observed
for C. minutissimum. The API Coryne system provided the numerical codes
1100325,
2100324, and 3100325 for C. imitans, indicating a negative α-glucosidase
reaction,
whereas all C. diphtheriaestrains express this enzyme. C.
imitans strains do not
produce
propionic acid as a fermentation product, unlikeC. diphtheriae (8),
and the CFA
composition
profiles for the species qualitatively differ, as C. diphtheriae and
closely related
species
have a unique pattern among Corynebacterium species. Diphtheria toxin
assessment
by
Elek testing or by assaying for the tox gene by PCR are negative for C.
imitans strains
(8, 49). C.
imitans is resistant to O/129, while C. diphtheriae is not.
C.
jeikeium
C.
jeikeium is a frequently encountered Corynebacterium in clinical
specimens (72).
Nosocomial
transmission has been described. The complete genome sequence of a C.
jeikeium
strain has been determined (147, 148),
indicating that the lipophilic phenotype
of C.
jeikeium originates from the absence of fatty acid synthase. C. jeikeium
is often
resistant
to multiple antibiotics (including penicillin and gentamicin) (42, 75),
but this cannot
be
used as a taxonomic characteristic because the phenotypically closely related C.
tuberculostearicum
may also demonstrate multidrug resistance. Quantitative DNA-DNA
hybridization
experiments have shown that C. jeikeium includes two genomospecies for
which
penicillin and gentamicin MICs are low, but as they could not otherwise be
differentiated
phenotypically from the resistant C. jeikeium strains, they were not
proposed
as
independent species (124). Colonies of C. jeikeium are tiny, low, entire, and
grayish
white.
C. jeikeium is a strict aerobe which may oxidatively produce acid from
glucose and
sometimes
from maltose but not from fructose (C. tuberculostearicum is positive
for acid
production
from fructose). The RapID CB Plus system correctly identifies C. jeikeium, as
does
the
API Coryne system if ancillary tests are used (67, 69).
C.
kroppenstedtii
C.
kroppenstedtii (23), a rather rarely recovered species, was originally recovered
from the
sputum
of a patient with pulmonary disease. Additional strains have been isolated from
lung
biopsy
specimens, sputum, a breast abscess, and patients with granulomatous lobular
mastitis
(8, 110). Colonies are grayish, translucent, slightly dry, and less than
0.5 mm in
diameter
after 24 h of incubation at 37°C. C. kroppenstedtii is lipophilic and is
one of the few
medically
relevant Corynebacterium species exhibiting esculinase activity. Other
biochemical
characteristics
are given in Table 3. API Codes of C. kroppenstedtii include 0101104,
2040104,
and 2040105 (8). It can be separated from C. durum, C. matruchotii, and C.
glucuronolyticum
by colony and Gram stain morphologies and from C.
glucuronolyticum by
its
negative CAMP reaction. The determination of the whole-genome sequence revealed
that
lipophilism
is the dominant feature involved in the pathogenicity of C. kroppenstedtii (149).
C.
lipophiloflavum
Initially,
C. lipophiloflavum (55) was represented by a
single strain which had been isolated
from
vaginal discharge from a patient with BV. Additional strains have now been described
from
blood cultures (G. Funke, R. Frodl, E. Falsen, C. Sproer, H.-P. Klenk, and S.
Stenger,
submitted
for publication). This species contains lipophilic strains, but the majority of
strains
are
nonlipophilic (Funke et al., submitted). The lipophilic strains have the same
biochemical
screening
pattern as C. urealyticum, except that they exhibit a strong yellow
pigment and
weaker
urease activity and slowly produce acid from glucose (Table
3). In contrast to
most
C. urealyticum strains, the C. lipophiloflavum strains isolated
were not multidrug
resistant.
The nonlipophilic strains have a biochemical profile similar to that of C.
falsenii but
do
not ferment galactose and trehalose (Funke et al., submitted).
C.
macginleyi
C.
macginleyi (128) has almost exclusively been isolated from eye specimens, whether
from
diseased
(62) or healthy conjunctiva. Colonies are typical for lipophilic
corynebacteria (see
above).
When grown on Tween 80-SBA plates (better growth is usually found on plates
supplemented
with 0.1% Tween 80 than on those supplemented with 1.0% Tween 80),
some
C. macginleyi strains exhibit a rose pigment which is not seen for any
other
lipophilic
Corynebacterium species. C. macginleyi is one of the very
few Corynebacterium
species not expressing pyrazinamidase activity (Table
3). Most strains
ferment
mannitol, while the majority of other corynebacteria are unable to do so. The
API
Coryne
system correctly identifies C. macginleyi (69).
Strains belonging to this species are
susceptible
to a broad spectrum of antibiotics (62),
but high-level fluoroquinolone resistance
has
been reported (35).
C.
massiliense
This
species was isolated from synovial fluid (96).
It does not produce acid from any of the
carbohydrates
tested in the differentiation of true corynebacteria (Table
3), but its cell wall
contains
TBSA.
C.
mastitidis-Like Organism
Bacteria
recovered from ocular specimens (patients with cataracts, diabetic retinopathy,
or
dry
eyes) were by 16S rRNA gene sequencing found to be closest (98.2% identity) to C.
mastitidis,
which to date has otherwise been found to cause sheep mastitis. In
contrast to C
macginleyi,
these strains are susceptible to the fluoroquinolones, but no other
information
about
these strains is extant (35).
C.
matruchotii
C.
matruchotii is thought to be a natural inhabitant of the oral cavity,
particularly on calculus
and
plaque deposits, and so has been much studied by oral microbiologists (115).
Otherwise,
it
is a very rare human pathogen. Microcolonies appear flat, filamentous, and
spider-like, but
macrocolonies
have a variable appearance (19). C. matruchotii demonstrates
a very unusual
appearance
by Gram staining in that so-called whip handles (i.e., filamentous bacteria
with a
single
short bacillus adjacent to the end of the filament that creates the illusion of
a whip)
are
observed (Fig. 1f). This microscopic presentation is consistent even when isolates
that
have
been preserved for many years in a culture collection are stained. It has been
demonstrated
that heterogeneity exists among C. matruchotii strains obtained from
international
culture collections and that some strains represented were misidentified C.
durum
isolates (115). C. matruchotiistrains are consistently negative for
galactose,
whereas
C. durum strains can be positive. The API Coryne system database does
not
contain
C. matruchotii; the numerical codes observed for C. matruchotii include
7000325,
7010325,
and 7050325.
C.
matruchotii-Like Strain
A C.
matruchotii-like strain is represented by a single strain, ATCC 43833 (115).
It was
deposited
in the ATCC as C. matruchotii, but it is a distinct species, as revealed
by dot blot
hybridization
and 16S rRNA gene sequencing data (GenBank accession no. AF260434).
Colonies
are the size of pinpoints to 0.1 mm in diameter and grayish white, with a
smooth,
nonadherent
texture. Biochemical screening reactions are similar to those of C.
minutissimum
except that strain ATCC 43833 exhibits esculinase activity in the
API Coryne
system,
with an API Coryne code of 2140325.
C.
minutissimum
C.
minutissimum is part of the normal human skin biota, and its historical
association with
erythrasma
is highly questionable (72). Colonies of C. minutissimum are whitish gray, shiny,
moist,
convex, and circular; they have entire edges and are about 1 to 1.5 mm in
diameter
after
24 h. Most of the colonies are creamy, but some may also have a sticky
consistency. C.
minutissimum
strains have a fermentative metabolism and produce acid from
sucrose
variably.
Very few C. minutissimum strains are also able to produce acid from
mannitol. The
API
Coryne system identifies C. minutissimum, with additional tests being
necessary for most
strains
(69). Many C. minutissimum strains are pyrrolidonyl
arylamidase positive. C.
minutissimum
strains exhibit DNase activity, nearly all strains hydrolyze
tyrosine, and a very
few
strains exhibit a positive CAMP reaction. Lactic and succinic acids are major
end products
of
glucose metabolism. Some isolates possess TBSA in their cell membranes. Nearly
all C.
minutissimum
strains are susceptible to O/129 (150-μg disk); i.e., they exhibit
an inhibition
zone
around the disk (usually between 20 and 35 mm in diameter). Phylogenetically, C.
minutissimum
is closely related (>98.8% identity) to both C. singulare and
C.
aurimucosum
by 16S rRNA gene sequencing, but these are readily discerned by
partial
rpoB gene sequence analysis.
C.
mucifaciens
C.
mucifaciens (58) has been isolated mainly from blood cultures and other sterile
body
fluids
but has also been recovered from abscesses, soft tissue, and dialysate (8).
Colonies
are
very distinct because they are slightly to overtly yellow and very mucoid (Fig.
3c) (very
few
strains are not mucoid [G. Funke, unpublished observation]). C. mucifaciens is
the only
presently
known Corynebacterium species exhibiting such mucoid colonies; this
characteristic
strongly
reminds bacteriologists of Rhodococcus equi colonies. An extracellular
substance
(probably
polysaccharides) causing connective filaments between the cells has been
demonstrated
as the ultrastructural correlate of the mucoid colonies. Colonies are about 1
to
1.5
mm after 24 h of incubation and have entire edges. They appear less mucoid
after
extended
incubation for 96 h. C. mucifaciens has an oxidative metabolism. It
consistently
produces
acid from glucose, but acid production from sucrose is variable. The API Coryne
numerical
codes 2000004, 2000104, 2000105, 2100104, 2100105, 6000004, 6100104, and
6100105
have been observed for C. mucifaciens, suggesting that occasionally
glucose
oxidation
may be too slow to be observed by that method. C. mucifaciens is
enzymatically
less
active than R. equi, which exhibits α- and β-glucosidase activities not
observed for C.
mucifaciens.
In addition, C. mucifaciens produces acid from fructose and
may produce acid
from
glycerol and mannose, but acid production from these sugars is not seen in R.
equi
strains. TBSA can be detected in amounts of 1 to 2% of the total CFAs.
β-Lactam
antibiotics
and aminoglycosides show very good activities against C. mucifaciens.
C.
pilbarense
This
nonlipophilic species was isolated from an ankle aspirate of a male thought to
be
suffering
from gout (3). It can be differentiated from C. striatum and C.
simulans by being
nitrate
reduction negative and from C. minutissimum by not fermenting maltose.
Phylogenetically,
it is closest (98.7 to 99.0% identity) to C. ureicelerivorans, C. coyleae, C
afermentans,
and C. mucifaciens but was distinguished from those species
by DNA-DNA
hybridization
and biochemically (Table 3) (3).
C.
propinquum
C.
propinquum is the closest phylogenetic relative of C. pseudodiphtheriticum
(108, 133) and
shares
the same niche (i.e., the oropharynx) as C. pseudodiphtheriticum. Colonies
are
whitish
and somewhat dryish, with entire edges, and are 1 to 2 mm in diameter after 24
h of
incubation.
This species reduces nitrate and hydrolyzes tyrosine but does not hydrolyze
urea
(Table
3). The API Coryne system and the RapID CB Plus system correctly
identify C.
propinquum
strains (67, 69).
C.
pseudodiphtheriticum
C.
pseudodiphtheriticum is part of the normal oropharyngeal biota. As described in Table
2,
this
species has been well documented to cause pneumonia in various patient
populations.
Colonies
are whitish and slightly dry, with entire edges, and they are 1 to 2 mm in
diameter
after
48 h of incubation. This nonfermenting species reduces nitrate and hydrolyzes
urea but
does
not produce acid from any of the commonly tested carbohydrates (Table
3). Some
strains
hydrolyze tyrosine. The API Coryne and the RapID CB Plus systems correctly
identify
C. pseudodiphtheriticum strains (67, 69).
Imperfectly cleaved mycolic acids coelute
with
CFAs (7). C. pseudodiphtheriticum strains are susceptible to
β-lactam antibiotics, but
resistance
to macrolides and lincosamides has been observed.
C.
pseudotuberculosis
C.
pseudotuberculosis is phylogenetically closest to C. ulcerans and to C.
diphtheriae
(108, 133); like those species, it may harbor the diphtheria toxin gene and
produce
propionic acid as a fermentation product, and its cell wall contains large
amounts of
the
CFA C16:1v7c (7). Colonies are yellowish white, opaque, convex, and about 1 mm in
diameter
after 24 h. Like C. ulcerans, C. pseudotuberculosis is positive for
urease and
positive
by the CAMP inhibition test. In this test, complete inhibition of the effect of
S.
aureus
beta-hemolysin on sheep erythrocytes is achieved by streaking the
presumed C.
pseudotuberculosis
strain in a right angle toward S. aureus and incubating
overnight; a betahemolysin
inhibition
zone in the form of a triangle is observed, as is true for A.
haemolyticum
(Fig. 2). C. pseudotuberculosis is not susceptible to O/129,
whereas C.
ulcerans
strains are. C. pseudotuberculosis has varied results for
both nitrate reduction and
sucrose
fermentation. The API Coryne system and the RapID CB Plus panel correctly
identify
this
species (67, 69). Human disease to date has been acquired by handling of infected
sheep.
C.
pyruviciproducens
This
species was independently isolated from a groin abscess (151)
and a urethral swab (G.
Funke,
unpublished observation) and was initially believed to be a lipophilic variant
of C.
glucuronolyticum,
which was β-glucuronidase and CAMP test positive. However, 16S
rRNA
gene
sequence analysis, quantitative DNA-DNA hybridization, and partial rpoB gene
sequence
analysis clearly demonstrated that this species is distinct fromC.
glucuronolyticum
(151).
C.
resistens
This
species has entire, grayish-white, and glistening colonies and is lipophilic.
It is unusual
in
having a negative pyrazinamidase reaction, which separates it from the
phenotypically
related
C. jeikeium or C. tuberculostearicum. In addition, C.
resistens grows slowly under
anaerobic
conditions, whereas C. jeikeium is unable to do so. The C. resistens strains
reported
in the literature are resistant to penicillin, cephalosporins, aminoglycosides,
clindamycin,
and ciprofloxacin but remain susceptible to glycopeptides (106).
It is presently
unknown
whether true C. resistens strains are often misidentified as C.
jeikeium in routine
laboratories.
C.
riegelii
C.
riegelii strains were originally described as being isolated from females
with urinary tract
infections
(59), but additional strains have been recovered from blood cultures,
including
cord
blood (8). Colonies are whitish, glistening, and convex, with entire
margins, and are up
to
1.5 mm in diameter after 48 h of incubation. Some colonies are of a creamy
consistency,
whereas
others are sticky. C. riegelii strains exhibit a very strong urease
activity with
Christen
sen’s urea broth, becoming positive within 5 min at room temperature after
inoculation.
A very peculiar characteristic of C. riegelii is the slow fermentation
of maltose
but
not glucose. No other definedCorynebacterium species exhibits this
feature (Table 3).
The
weak anaerobic growth of C. riegelii corresponds to the weak
fermentative metabolism.
API
Coryne system codes observed for C. riegelii include 0101224, 2001224,
and 2101224.
C.
simulans
This
species was originally delineated from some C. s triatum-like strains (159).
The three
strains
described in the original publication came from skin-related specimens (foot
abscess,
lymph
node biopsy specimen, and boil). Two additional strains have been
characterized, one
from
bile and one from a blood culture (8).
Colonies of C. simulans (grayish white, glistening,
creamy,
1 to 2 mm in diameter) are very similar to those of C. minutissimum, C.
singulare,
and C. striatum, its closest phylogenetic neighbors. C.
simulans is the only
validCorynebacterium
species described to date which reduces nitrite. Further characteristics
which
separate C. simulans from the closely related nonlipophilic,
fermentative
corynebacteria
are an inability to acidify ethylene glycol and to grow at 20°C (unlike C.
striatum).
API Coryne profiles include 0100305, 2100105, 2100301, 2100305, and 3000125
(including
a falsely negative nitrate reduction reaction because of the strong nitrite
reduction).
C.
singulare
C.
singulare colonies are circular and slightly convex, with entire margins,
and are of a
creamy
consistency, as observed for C. minutissimum and C. striatum (130).
Key
biochemical
reactions are like those for C. minutissimum except that urease activity
is
observed
(Table 3). The numerical API Coryne system profile is 6101125, which
indicates
that
pyrrolidonyl arylamidase activity is present. Like C. minutissimum and C.
striatum, C.
singulare
hydrolyzes tyrosine. C. singulare does not produce
propionic acid as a fermentation
product,
differentiating it from C. amycolatum. Phylogenetically, this species is
closely
related
(>98.8% identity) to both C. aurimucosum and C. minutissimum by
16S rRNA gene
sequencing
but can readily be discerned by partial rpoB gene sequence analysis.
C.
sputi
This
species is the only true Corynebacterium that expresses α-glucosidase
activity and is
positive
for TBSA (164). It can be differentiated from C. ulcerans by being
positive for
pyrazinamidase
activity but negative for alkaline phosphatase and maltose fermentation.
Unlike
C. ulcerans, this species cannot harbor the bacteriophage which bears
the
diphtheria
tox gene.
C.
stationis
This
species, originally called Brevibacterium stationis, has recently been reassigned
to the
genusCorynebacterium
(9). B. stationis ATCC 14403T was originally recovered from
sea
water,
but subsequent studies found that two human blood culture isolates plus “C.
ammoniagenes”
ATCC 6872, recovered from an infant’s stools, formed a single
taxon group
now
designated C. stationis comb. nov. Strains of both C. stationisand
C.
ammoniagenes
sensu stricto demonstrate the ability to alkalinize citrate, using
either
Simmon’s
citrate or a heavy (not light) inoculum in the citrate reaction chamber found
in an
API
20E strip, a feature not previously associated with members of the
genus
Corynebacterium. Colonies of C. stationis grow well in 24 h (that
is, they are not
lipophilic),
are yellow or yellowish, and ferment glucose, fructose, ribose, and mannose.
All
are
positive for the alkalinization of citrate, produce urease, reduce nitrate, and
hydrolyze
tyrosine
(other features are shown in Table 3). API Coryne codes
generated were 1001304
and
3001304 (that is, the enzyme PYZ is variably detected). Strains of this species
are
susceptible
to all antimicrobials; however, one blood culture isolate was shown to be
resistant
to erythromycin.
C.
striatum
C.
striatum is part of the normal human skin biota. Nosocomial transmission of
C.
striatum
has been documented (89, 121).
Colonies are convex, circular, shiny, moist, and
creamy,
with entire edges, and are about 1 to 1.5 mm in diameter after 24 h of
incubation.
Some
investigators have described C. striatum colonies as being somewhat like
those of
small
coagulase-negative staphylococci. C. striatum has a fermentative
metabolism, and acid
production
from sucrose is variable. The API Coryne system identifies C. striatum,
but
additional
tests are needed in most cases (69).
All C. striatum strains hydrolyze tyrosine,
and
some strains are CAMP reaction positive; however, the CAMP reaction of C.
striatum
strains is usually not as strong as that of other CAMP
test-positive species (e.g., C.
auris
or C. glucuronolyticum). Lactic and succinic acids are the
major end products of glucose
metabolism.
All C. striatum strains are susceptible to O/129. Resistance to
macrolides and
lincosamides
due to the presence of an rRNA methylase has been described. C. striatum may
also
be resistant to quinolones and tetracyclines (42, 75),
which has led to a renewed
interest
in this agent as an emerging pathogen (10).
C.
sundsvallense
C.
sundsvallense (8, 20) has been isolated from blood cultures, a vaginal swab, and a
sinus
drainage
specimen from an infected groin. Colonies of this nonlipophilic species are
buff or
slightly
yellowish, they adhere to agar, and they have a sticky consistency. Gram
staining
shows
bulges or knobs at the ends of some rods, and these are not seen in any other
corynebacteria.
Fermentation of glucose, lactose, and sucrose is slow (Table
3). C.
sundsvallense
can be separated from C. durum by its positive
α-glucosidase reaction and its
inability
to ferment galactose. It is differentiated from C. matruchotii by
expressing urease
but
not nitrate reductase activity and by not producing propionic acid as an end
product of
glucose
metabolism (8, 20).
C.
thomssenii
C.
thomssenii (169) is a rarely found species. It was originally repeatedly isolated
from a
patient
with pleural effusion, and a second strain was recovered from the environment
in
Canada
(8). This species is fastidious and grows slowly, resulting in
colonies of <0.5 mm
after
48 h, but it is not lipophilic. After 96 h, colonies are molar-tooth-like, very
sticky, and
slightly
adherent to agar. The clinical strain of C. thomssenii is the onlyCorynebacterium
species
expressing N-acetyl-β-glucosaminidase activity, which can be observed in
either the
API
Coryne or the API ZYM system. Acid is slowly and fermentatively produced from
glucose,
maltose,
and sucrose, and the resulting API Coryne code for C. thomssenii is
2121125.
C.
timonense
This
species was isolated from blood cultures of a patient with endocarditis. Its
most peculiar
feature
is a weakly positive esculinase reaction. It can be differentiated from other
esculinase-positive
corynebacteria by having negative reactions for maltose and sucrose
fermentation
as well as a negative CAMP reaction (96).
C.
tuberculostearicum
This
species was revived for a never validly published taxon and also includes a
strain of the
nonvalidated
species “C. pseudogenitalium” (43).
Nearly all “CDC group G” bacteria can be
assigned
to C. tuberculostearicum(K. A. Bernard and G. Funke, unpublished
observation).
Unfortunately,
the effective publication on C. tuberculostearicum did not include any
strains
of
CDC group G bacteria (43). C. tuberculostearicum has some variable biochemical key
reactions
and can be differentiated from C. accolens if TBSA is detected as one of
its CFAs. It
can
be separated from C. jeikeium by anaerobic growth and fermentative acid
production
from
fructose. C. tuberculostearicum can be multidrug resistant, but the most
frequently
observed
resistance is to macrolides and lincosamides.
C.
tuscaniense
This
species has been isolated from blood cultures of a patient suffering from
endocarditis. C.
tuscaniensedoes
not grow under anaerobic conditions, which distinguishes this species from
the
phenotypically similar C. minutissimum. In addition, C. tuscaniense hydrolyzes
hippurate
but
not tyrosine, whereas C. minutissimumhas opposite reactions (Table
3). C.
tuscaniense
colonies are rounded and regular, in contrast to the biochemically
similar
species
C. amycolatum, whose colonies exhibit irregular edges (123).
C.
ulcerans
Phylogenetically,
C. ulcerans (129) is closely related to C. pseudotuberculosis, and both
species
are the closest relatives to C. diphtheriae (108, 133).
Its cell membrane contains
significant
amounts of the CFA C16:1v7c. As described previously, C. ulcerans can
harbor the
diphtheria
toxin gene, but differences in the receptor-binding and translocation domains
have
been described, so a C. ulcerans diphtheria toxin-specific PCR has been
developed for
conventional
PCR (138) or real-time PCR (15, 135, 136). Disease associated with this
bacterium
is rare, but if it is recovered from pseudomembranous material, it must be
treated
like
a case of diphtheria (33,34). Recent reports have described the transmission of C.
ulcerans
from companion pets to humans (11, 31,88),
and so expanded national cases of
diphtheria-like
diseases that include cases involving toxigenic strains of C. ulcerans or
C.
pseudotuberculosis
have formally been defined in some countries (K. Bernard,
unpublished
observation)
(11). C. ulcerans colonies are somewhat dry, waxy, and grayish
white, with
light
hemolysis; they are 1 to 2 mm in diameter after 24 h. C. ulcerans may be
differentiated
from
C. diphtheriae by urease activity and a CAMP inhibition reaction and
from C.
pseudotuberculosis
by source, biochemically, and by partial rpoBgene but not
16S rRNA gene
sequencing.
Strains of C. ulcerans are positive for glycogen, starch, and trehalose
fermentation.
The API Coryne system and the RapID CB Plus identification strip correctly
identify
C. ulcerans(67, 69).
C.
urealyticum
C.
urealyticum is one of the relatively frequently isolated clinically
significant corynebacteria
in
clinical specimens (72). C. urealyticum is strongly associated with
urinary tract infections.
Recovery
of this bacterium is often associated with urine with an alkaline pH, resulting
in
struvite
crystals. As for all other lipophilic corynebacteria, colonies are the size of
a pinpoint,
convex,
smooth, and whitish gray on regular SBA. C. urealyticum is a strict
aerobe and has
very
strong urease activity (Table 3). Commercial identification systems correctly identify C.
urealyticum.
C. urealyticum is almost always multidrug resistant (42, 72, 140).
Sequencing
of
the whole C. urealyticum genome indicated that the multidrug resistance
is mediated by
transposable
elements (150).
C.
ureicelerivorans
Lipophilic
C. ureicelerivorans, first described from a blood culture, has very
strong and
rapidly
detected (~60 s) urease activity as its most prominent feature (163).
Subsequently,
recovery
of this organism from ascites fluid and multiple blood cultures from
immunocompromised
patients or those with digestive disorders has been described (41).
It
can
be differentiated from CDC group F-1 bacteria by a positive alkaline
phosphatase
reaction
and by a negative reaction for acid production from maltose and sucrose. By 16S
rRNA
and partialrpoB gene sequencing, this species is closest to C.
mucifaciens; C.
ureicelerivorans
can readily be discerned from that species by its smooth rather than
mucoid
yellowish
colonies as well as by observation of rapid urease activity.
C.
xerosis
C.
xerosis colonies are dry, granular, and yellowish, with irregular edges,
and are 1 to 1.5
mm
in diameter after 24 h. It must be emphasized that nearly all “C. xerosis” strains
which
have
been described in the literature before 1996 may have been misidentified C.
amycolatum
strains (56). C. striatum strains were also misidentified as C.
xerosis in the
past.
C. xerosis has a fermentative metabolism and has variable results for
the presence of
nitrate
reductase but always expresses α-glucosidase as well as leucine arylamidase
activity.
Because
C. xerosis was thought to be rarely encountered in clinical specimens,
it was not
included
in the API Coryne system version 3.0 database. The numerical profiles observed
for C.
xerosis strains, such as 2110325 and 3110325, provide API Web responses
that
include
“C. striatum/C. amycolatum.” The RapID CB Plus system correctly
identifies C.
xerosis
(67). Lactic acid is the major end product of glucose metabolism, and
strains are
susceptible
to O/129. As described previously, C. xerosis is closely
phylogenetically related
to C.
freneyi and C. hansenii (119),
and so if definitive identification is required,
characterization
must include genetic testing.
Genus Turicella
T.
otitidis is almost exclusively isolated from clinical specimens from the
ear region, but it
does
not cause otitis media with effusion in children. Colonies are whitish, convex,
and
creamy,
with entire edges, and are 1 to 1.5 mm in diameter after 48 h of incubation.
Some
young
colonies show a greenish appearance when taken away from the plates with a
swab.
The
distinctive Gram stain morphology of T. otitidis is given in Fig.1b.
Differentiation from C.
auris
and C. afermentans subsp. afermentans is readily
achieved by morphologic features,
but
utilization reactions may also assist in the differentiation of these taxa (Tables
3 and 4)
(57,118).
All T. otitidis strains are strongly CAMP reaction positive and give the
numerical
code
2100004 in the API Coryne system. The MICs of β-lactam antibiotics for many
strains
are
very low; some strains might be resistant to macrolides and clindamycin (68, 75).
The
mechanism
of resistance to macrolides appears to be due in part to mutations in the 23S
rRNA
(rrl) gene (12).
Genus Arthrobacter
Arthrobacter
spp. might be part of the indigenous normal human biota, but their
main
habitat
is soil. A. cumminsii seems to be a normal commensal in humans and
appears to be
the
most frequently isolatedArthrobacter species in human clinical specimens
(63, 92),
and A.
oxydans is the second-most-frequently encountered species
(92). Arthrobacter
colonies are usually whitish gray, slightly glistening, creamy, and 2 mm or
greater
in diameter after 24 h. A. cumminsii is slightly smaller than the other
arthrobacters
and
may also exhibit a sticky consistency (63). Arthrobacter
spp. usually do not oxidize any
of
the carbohydrates routinely tested and do not emit a cheese-like smell, as is
often found
for
the phenotypically closely related brevibacteria. Some arthrobacters are
motile, whereas
brevibacteria
are always nonmotile. Like brevibacteria,Arthrobacter spp. express DNase
and
have
gelatinase activity (54). The identification of arthrobacters to the species level might
be
achieved
by carbohydrate utilization tests, but this is recommended for reference
laboratories
only. A. albus (160) is phylogenetically most closely related to A. cumminsii but
might
be differentiated phenotypically by being resistant to desferrioxamine, whereas
A.
cumminsii
is susceptible. A. cumminsii has a distinctive CFA pattern,
with C14:0i and
C14:0
each representing 2 to 4% of all CFAs (54).
Penicillin MICs for most Arthrobacter strains
are
low, with quinolones showing only weak activities againstArthrobacter spp.
(54, 92).
Genus Brevibacterium
Some
Brevibacterium spp. are part of the normal human skin biota. Colonies
are whitish
gray
(or yellowish likeB. luteolum), convex, mostly creamy, and 2 mm or
greater in diameter
after
24 h. B. mcbrellneri colonies have a more granular appearance and are
dryer than
those
of other brevibacteria. Some brevibacteria may develop a yellowish or greenish
pigment
after prolonged incubation. Many Brevibacterium strains isolated from
human
clinical
material give off a distinctive cheese-like odor. Brevibacteria are nonmotile,
are
halotolerant
(6.5% NaCl), and form methanethiol from methionine, but this test is specific
for
brevibacteria only when it is read within 2 h (48).
Brevibacteria can be identified to the
species
level by carbohydrate utilization tests. More than 90% of all
clinical
Brevibacterium isolates are B. casei (48). Brevibacterium
sanguinis is very similar
to B.
caseiand can be differentiated from this species by susceptibility to
thallium acetate.
The
MICs of β-lactam antibiotics for brevibacteria are often elevated (68).
Genus Dermabacter
D.
hominis strains are part of the normal skin biota. Colonies are whitish,
convex, of a
creamy
or sticky consistency, and 1 to 1.5 mm in diameter after 48 h (Fig.
3e). D.
hominis
strains are sometimes mistaken for small-colony coagulase-negative
staphylococci.
The
Gram staining result is distinctive, with coccobacillary or coccoidal forms (Fig.
1c). The
key
biochemical reactions are given in Table 4. D.
hominis is one of the few coryneform
bacteria
with a variable reaction for xylose fermentation. It is the only
catalase-positive
coryneform
bacterium (except Actinomyces neuii) that is able to decarboxylate
lysine and
ornithine
(70). The API Coryne system and the RapID CB Plus panel correctly
identify this
species (67, 69). D.
hominis strains may be resistant to aminoglycosides (68, 152).
TYPING SYSTEMS Back to top
Outbreaks of C. diphtheriae in the states
of the former Soviet Union and other locations have
been studied by whole-cell peptide analysis,
whole-genome restriction fragment length
polymorphism analysis, ribotyping, pulsed-field
gel electrophoresis, PCR–single-strand
conformation polymorphism analysis, analysis of tox
anddtxR as well as of the 16S-23S rRNA
spacer region, amplified fragment length
polymorphism analysis, random amplification of
polymorphic DNA, and multilocus enzyme
electrophoresis (30, 113). An international
database for C. diphtheriae ribotypes using
the endonuclease BstEII has been established
(76). Ribotyping is regarded as being highly
discriminatory and, based on a comprehensive
comparison of methods, was found to be a preferred
typing method for C. diphtheriae (30).
Other methods, including a spoligotyping system
(similar to the spacer oligonucleotide typing
for Mycobacterium tuberculosis), have been
described (98). Sequencing studies with C.
diphtheriae strains from the epidemic in the former Soviet Union have shown
that point
mutations within the tox gene were silent
mutations and that multiple point mutations
(which even led to amino acid substitutions) were
observed for the dtxR gene, corresponding
to the heterogeneity of outbreak strains as
revealed by PCR–single-strand conformation
polymorphism analysis (100).
Isolates derived from specific populations in the United States
and Canada and characterized by multilocus enzyme
electrophoresis, ribotyping, and random
amplification of polymorphic DNA were found to be
members of persistent endemic strains,
rather than being imported from other countries
where diphtheria is endemic. More recently,
a standardized multilocus sequence typing method
based on analysis of short sequences
derived from seven housekeeping alleles has been
under development, with which
concatenated sequence data, sent by the Internet
to the curator (initially the University of
Warwick and then the Institut Pasteur, Paris
[relocation in 2009]), are compared to a large
database and given an existing or new sequence
type (25). Due to the technical simplicity of
this approach and the ease of comparison of strain
data internationally, this is being touted
as a typing method for the future. Multilocus
sequence typing has also been effectively
applied to an outbreak investigation involving C.
macginleyi and C mastitidis-like isolates
(35).
SEROLOGIC TESTS Back to top
Detection of antibodies directed against
diphtheria toxin is the only established serologic test
for coryneform bacteria. Toxin neutralization
assays using a Vero cell culture system have
been replaced mainly by enzyme immunoassays.
Levels of ≥0.1 IU/ml serum are thought to
confer protection, whereas levels of <0.01
IU/ml indicate a susceptible host and levels of
0.01 to 0.1 IU/ml indicate partially immune
individuals. It is believed that between 20 and
60% of adults in the United States lack protective
antibodies to diphtheria toxin due to
declining antibody titers in immunized persons and
in those persons who did not receive the
primary immunization series. This could pose a
potentially significant public health risk and
could result in the reemergence of this disease.
Booster doses of toxoid should be
administered at 10-year intervals.
ANTIMICROBIAL SUSCEPTIBILITIES Back to top
The susceptibility patterns for each taxon were
given with the descriptions of each taxon (see
above). Since the antimicrobial susceptibility of
coryneform bacteria is not predictable in
every case, susceptibility testing should always
be performed with clinically significant
isolates (see “Clinical Significance” above). Due
to the emergence of vancomycin-resistant
gram-positive organisms, it has become
inappropriate to recommend glycopeptides as firstline
drugs for the treatment of infections caused by
coryneform bacteria. It is also
noteworthy that some coryneform bacteria (e.g., Microbacterium
resistens) are intrinsically
vancomycin resistant.
The Clinical and Laboratory Standards Institute
has published testing conditions and
interpretive criteria for susceptibility testing
of coryneform bacteria using a broth
microdilution method (18).
Direct colony suspensions equivalent to a 0.5 McFarland standard
are prepared and strains are incubated in
cation-adjusted Mueller-Hinton broth with 2 to 5%
(vol/vol) lysed horse blood at 35°C in ambient air
for up to 48 h. Interpretative categories
for the MICs obtained are presently available for
16 antimicrobial agents. In summary, using
the broth microdilution method, it has recently
been established that multidrug resistance
was found for 4 or more drug classes (out of 16
classes tested) for some or most strains
of C. amycolatum, C. jeikeium, C. urealyticum,
C. resistens, C. tuberculostearicum, and C.
striatum, as well as representatives of C. afermentans and C.
aurimucosum found in one
national culture collection (C. Singh, T. Burdz,
and K. Bernard, unpublished observations).
Very few studies have been performed comparing
broth microdilution and disk diffusion
results for susceptibility testing of coryneform
bacteria (162).
In the past, MICs were determined by either the
Etest or the agar dilution or broth
microdilution method. The results of the Etest
have been shown to correlate reasonably well
with those of both the broth microdilution and the
agar dilution method
for Corynebacterium spp. (95,
167). The Etest should be carried out on
Mueller-Hinton agar
supplemented with 5% sheep blood. The same medium
is used for the agar dilution method
(68), but this method is not applicable in routine
laboratories and, rather, should be used in
studies with individual antimicrobial agents.
Metronidazole is the drug of choice both for local
therapy of BV and for systemic therapy of
extravaginal infections caused by BV-associated
biota. Systemic infections due to G.
vaginalis alone can be treated with ampicillin or amoxicillin, since
β-lactamase-producing G.
vaginalis strains have not been observed so far. Susceptibility testing for G.
vaginalis is not
recommended.
EVALUATION, INTERPRETATION, AND REPORTING OF
RESULTS Back to top
The guidelines related to when coryneform bacteria
should be identified to the species level
(see “Clinical Significance” above) are also
applicable for evaluating and interpreting culture
results; i.e., whenever coryneform bacteria are
identified to the species level, the results
should be reported.
In the rare case of microscopically suspected C.
diphtheriae (i.e., a positive Neisser staining
result), the physician in charge of the patient
should be notified immediately, although
culture results and toxin testing results become
available only later.
It is evident that repeated isolation of a
predominant strain of a coryneform bacterium or a
coryneform bacterium growing in pure culture
suggests an etiological relationship to the
patient’s disease. If coryneform bacteria are
present in blood cultures, the physician in
charge should be notified immediately, and it
should be emphasized when reporting that the
clinical significance of the coryneform bacteria
must be carefully examined by cooperation
between the microbiology laboratory and the
physician. In our experience, one positive blood
culture out of two or three aerobically and
anaerobically incubated pairs of blood cultures is
hardly ever clinically significant (except in
cases of treated endocarditis). Care must be taken
in the interpretation of the results for those
patients for whom half or more of the blood
specimens taken for culture become positive for
coryneform bacteria, in particular when
lipophilic corynebacteria are cultured, since not
all blood samples taken from patients with
endocarditis due to lipophilic corynebacteria may
eventually become positive.
On the other hand, coryneform bacteria should be
reported as “normal flora” when they are
grown from nonsterile sites together with other
resident biota in equal or smaller numbers. It
is suggested that the primary isolation plates be
retained for at least 72 h before they are
discarded in order to have the opportunity to assess the bacterial
population retrospectively.
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