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Journal of Clinical Microbiology, December 1998, p. 3698-3702, Vol. 36, No. 12
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Differentiation of Corynebacterium amycolatum,
C. minutissimum, and C. striatum by
Carbon Substrate Assimilation Tests
François N. R.
Renaud,1,2,*
Marianne
Dutaur,3
Salah
Daoud,1
Dominique
Aubel,2
Philippe
Riegel,4
Daniel
Monget,5 and
Jean
Freney1,3
DERBA UPRES EA 1655, Faculté de
Médecine RTH Laennec, 69372 Lyon,1
Institut des Sciences Pharmaceutiques et Biologiques
(ISPB), 69373 Lyon,3
IUT A Lyon 1, 69622 Villeurbanne Cedex,2
Institut de
Bactériologie, Université Louis Pasteur, 67000 Strasbourg,4 and
bioMérieux, 38390 La Balme les Grottes,5 France
Received 21 May 1998/Returned for modification 2 July 1998/Accepted 10 September 1998
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ABSTRACT |
We tested the carbon substrate assimilation patterns of 40 Corynebacterium amycolatum strains, 19 C. minutissimum strains, 50 C. striatum strains, and 1 C. xerosis strain with the Biotype 100 system
(bioMérieux, Marcy-l'Étoile, France). Twelve carbon substrates of 99 allowed discrimination among the species tested. Additionally, assimilation of 3 of these 12 carbon substrates (maltose,
N-acetyl-D-glucosamine, and phenylacetate) was
tested with the API 20 NE identification system (bioMérieux).
Since concordant results were observed with the two systems for these three carbon substrates, either identification system can be used as a
supplementary tool to achieve phenotypic differential identification of
C. amycolatum, C. minutissimum, and C. striatum in the clinical microbiology laboratory.
| |
TEXT |
Recent progress in molecular
taxonomy (DNA-DNA hybridization and 16S rRNA sequencing) and in
chemotaxonomy has profoundly modified the classification of coryneform
bacteria. Since 1987, 24 former CDC groups have been assigned a new
genus and/or species name (8). Corynebacterium
amycolatum, C. minutissimum, and C. striatum
are frequently encountered in the routine clinical microbiology
laboratory (11, 15). Their normal habitat is the human skin
and mucous membranes, and they are therefore sometimes isolated as
contaminants in clinical samples. However, they have also been reported
to be responsible for various types of infection such as pneumonia,
endocarditis, and septicemia, especially in immunocompromised patients
(8, 11). Consequently, they should not always be considered
contaminants and should be identified to the species level. In
published case reports, C. amycolatum (13) and
C. striatum (3, 10, 12, 14, 16, 20) were all
found to be responsible for infection. However, differential identification of these three species by biochemical tests remains difficult, and several misidentifications have been reported previously (7, 8, 21, 23). Furthermore, interpretation of the clinical importance of these species is still difficult. These species have been
easily differentiated by methods that cannot easily be used in the
routine laboratory, such as chromatography of mycolic acids
(2), determination of propionic acid and lactic acid production by gas-liquid chromatography (5, 21), amplified ribosomal DNA restriction analysis (18), and amplification
of the 16S-23S gene spacer regions (1). Identification
schemes which simplify correct identification in the routine laboratory have recently been reported (8, 15, 19). Additionally, four
new tests which allow convenient differentiation of C. amycolatum, C. minutissimum, and C. striatum
have recently been established by Wauters et al. (22).
We report here on a study of carbon substrate assimilation by 110 strains belonging to these three Corynebacterium species and
conclude with a simple scheme allowing identification of C. amycolatum, C. minutissimum, and C. striatum
in the routine microbiology laboratory.
Bacterial strains.
We tested 110 Corynebacterium
strains isolated from various clinical samples from nonduplicate
patients. They were obtained from the bacterial collections of the
Département d'Étude et de Recherche en Bactériologie
Médicale (Lyon, France), IUT A Lyon 1 (Lyon, France),
bioMérieux Laboratories (La Balme-les-Grottes, France), and
the Microbiology Laboratory, Faculty of Medicine (Strasbourg, France).
In preparation for this study, these strains were identified to the
species level: C. amycolatum (40 strains), C. minutissimum (19 strains), C. striatum (50 strains),
and C. xerosis (1 strain) by recently described methods
(1, 8, 15, 19).
In brief, as a first step we inoculated API Coryne systems with these
strains (bioMérieux, Marcy l'Étoile,
France). The interpretations of the results were based on the
second-generation database (9). In addition to the API
Coryne system, we also determined the strains' capability for growth
under anaerobic conditions and in the absence of lipids and tested them
for the presence of a tyrosinase by previously described protocols
(15). In cases of ambiguity, the identification was
confirmed by PCR-based amplification of the 16S-23S gene spacer region
recently described by Aubel et al. (1). Identification of
C. amycolatum strains was confirmed by the absence of
mycolic acids according to the method described by Barreau et al.
(2). Additionally, we tested the following reference
strains: C. amycolatum CIP 103452T (Collection
de l'Institut Pasteur, Paris, France), C. minutissimum ATCC
23348T, C. striatum ATCC 6940T, and
C. xerosis ATCC 373T (American Type Culture
Collection, Manassas, Va.).
Culture conditions.
Corynebacterium strains were grown
at 37°C for 48 h on Columbia agar supplemented with 5%
(vol/vol) sheep blood (bioMérieux) in an atmosphere containing
10% CO2.
Carbon substrate assimilation tests. (i) Biotype 100 system
(bioMérieux).
The system is composed of 99 test wells, each
one containing a single dehydrated carbohydrate, organic acid, or amino
acid, plus one control well without carbon substrate. As a minimal
growth medium, we used Biotype Medium 2 (bioMérieux), which
contains 31 growth factors and is therefore adapted to fastidious
microorganisms. A bacterial suspension was prepared in 5 ml of
distilled water and adjusted to the density of a 6.0 McFarland
standard. Two milliliters of this suspension was transferred in 60 ml
of Biotype Medium 2, and this final suspension was used to inoculate
the system's wells. The inoculated system was incubated at 30°C in a
humid chamber. Growth was indicated by a higher turbidity observed in the test well than in the control well. In contrast to what is recommended for members of the family Enterobacteriaceae by
the manufacturer, only growth (and not color development) was recorded in test wells 19 (esculin), 39 (hydroxyquinoline 
-glucuronide), 59 (L-tryptophan), and 79 (L-histidine). Test
results for the 99 wells were recorded with the Recognizer software
package (P. A. D. Grimont, Taxolab, Institut Pasteur). The
interstrain distances were calculated by using the complement of the
Jaccard coefficient, which is not able to score double-negative
characteristics. Clusters were formed with the unweighted pair group
method with average (Taxotron package; Taxolab).
(ii) API 20 NE system (bioMérieux).
This system is
commercialized for the identification of gram-negative bacilli. To
adjust it to coryneform bacteria, we modified the manufacturer's
inoculation protocol by using a bacterial suspension adjusted to the
density of a 6.0 McFarland standard. Ten drops of this suspension was
then transferred in the AUX medium provided with the system, and this
final suspension was used to inoculate the system's auxanogram wells.
The inoculated system was incubated at 30°C, and growth in the
maltose, N-acetyl-D-glucosamine, and phenylacetate wells was observed after 2 and 4 days.
Results and discussion.
Use of the API Coryne system presents
several problems, including difficulties in reading some enzymatic
reactions, absence of C. amycolatum from the database, and
often the need for supplementary tests to identify the two other
species (6). The new API Coryne system database includes
C. amycolatum, and C. xerosis is no longer included in this database (9). C. amycolatum,
C. minutissimum, and C. striatum give the same
code: (2-3)100(1-3)(0-2)(4-5). Certain C. striatum strains
give a code such as 3100115; furthermore, a few C. amycolatum strains are urease positive (4).
The results obtained with the Biotype 100 system are reported in Table
1. Among the 99 carbon substrates tested,
only 32 gave more than 20% positive results for at least one of the
species tested (Table 1). C. amycolatum, C. minutissimum, and C. striatum used 13, 28, and 26 different substrates as sole carbon source, respectively. In general,
we find that the metabolic activity of C. amycolatum is much
lower than that of the two other species. The more discriminating
carbon substrates were D-galactose, maltotriose, maltose, N-acetyl-D-glucosamine,
phenylacetate, 4-aminobutyrate, 5-aminovalerate,
L-glutamate, D-alanine, L-alanine,
L-serine, and L-tyrosine.
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TABLE 1.
Percentages of positive carbon substrate assimilation
reactions for C. amycolatum, C. minutissimum,
and C. striatuma
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|
In our study, the dendrogram performed on the results of 99 carbon
substrate assimilation tests clearly showed three clusters
representing
the three species:
C. amycolatum,
C. minutissimum,
and
C. striatum (Fig.
1). However, the reference strain for
C. xerosis, ATCC 373
T, was included in the
C. amycolatum cluster, which we do not consider
a major
problem given the extremely rare occurrence of
C. xerosis in
clinical samples.
C. xerosis has been found only once in 750
isolates as reported by Wauters et al. (
22) and was
completely
absent in all of the 415 human isolates described by Riegel
et
al. (
15). Table
1 clearly shows that we can distinguish
all
three
Corynebacterium species by using the Biotype 100 system
under the above-mentioned conditions.
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FIG. 1.
Dendrogram of hierarchical aggregation clustering of 110 Corynebacterium strains belonging to the species C. amycolatum, C. minutissimum, C. striatum,
and C. xerosis (99 substrates of Biotype 100).
|
|
To facilitate the use of carbon substrate assimilation tests in the
routine clinical microbiology laboratory, we selected
the most
discriminating tests, i.e., maltose,
N-acetyl-
D-glucosamine,
and phenylacetate,
among those present in the API 20 NE system,
a commonly used carbon
substrate assimilation system. Since the
system is commercialized for
use with gram-negative bacilli, we
modified the manufacturer's
protocol to adapt the system to coryneform
bacteria and used a much
denser bacterial suspension as an inoculum.
Under these conditions, we
observed a good correlation between
the assimilation results observed
with the two systems, and the
use of only these three tests allowed a
simple and reliable differential
identification of the three species
(Table
1). However, there
exists an ambiguity in the maltose
assimilation of
C. amycolatum (66% positive scores by the
Biotype 100 system compared to 82%
by the API 20 NE system). This
difference could be explained by
the relatively higher inoculum used
for the API 20 NE system (0.5
ml of a 6.0-McFarland standard suspension
in 7 ml of minimal medium)
than for the Biotype 100 system (2 ml of the
same suspension in
60 ml of minimal medium). However, the different
inocula are expected
to play only a minor role since no difference
between the systems
could be detected for the assimilation of other
substrates. The
difference in maltose assimilation may therefore reside
rather
in different substrate concentrations found in the test chambers
of the two diagnostic
systems.
Since they are all irregularly shaped gram-positive coryneform
bacteria, it is very difficult to differentiate these species
by their
microscopic characteristics. However, the aspect, shape,
size, and
color of the colonies provide the clinical microbiologist
with useful
identification characteristics. After 24 h of incubation,
C. amycolatum produces characteristic dry colonies with an irregular
margin and a diameter of 0.5 mm. The colonies have a diameter
of 1 to
1.5 mm after 48 h of incubation and 2 mm after 72 h.
C. minutissimum colonies are smooth, convex, and shiny, and their
diameter varies from 1 to 1.5 mm after 24 h to 2.5 to 3 mm after
72 h of incubation.
C. striatum colonies are round,
regular, and
smooth (somewhat like coagulase-negative staphylococci)
after
24 h and measure between 2 and 3 mm after 72 h of
incubation.
The colony morphologies and sizes of all three species are
identical
when they are grown on blood-supplemented Trypticase soy agar
or on Columbia agar, except for
C. amycolatum, the colony
size
of which appears slightly smaller on Trypticase soy agar (about
0.5
mm).
Resistance to antibiotics, in particular ampicillin, could represent a
further diagnostic feature:
C. amycolatum is relatively
resistant to antibiotics, with one strain of two being resistant
to
ampicillin (
8,
15,
17). This represents a different
characteristic from
C. striatum and
C. minutissimum, most strains
of which are susceptible to this
antibiotic.
In summary, routine identification of catalase-positive, nonlipophilic,
coryneform gram-positive bacilli with the code (2-3)100(1-3)(0-2)(4-5)
when the API Coryne system is used can be performed according
to the
following scheme. (i) If the colony is rather dry with
an irregular
margin, it is
C. amycolatum. Confirmation will be
obtained
by resistance to ampicillin (one of two strains) and
phenylacetate and
N-acetyl-
D-glucosamine assimilations as tested
in the API 20 NE system, which will remain negative despite positive
maltose assimilation. (ii) If the colony is moist, convex, and
large
after 72 h of incubation and is nitrate reductase negative
and
maltose positive, it is
C. minutissimum. Confirmation is
obtained
by assimilation of the three substrates in the API 20 NE
system
and susceptibility to ampicillin. (iii) If the colony is moist,
with a diameter not larger than 1 mm after 48 h of incubation
but
reaching 2.5 to 3 mm after 72 h, and is nitrate reductase
positive
and maltose negative, it is
C. striatum. Confirmation
will
be obtained by assimilation of phenylacetate and absence
of
assimilation of maltose and
N-acetyl-
D-glucosamine in the API
20 NE system.
Apart from a few exceptions, the strain is sensitive
to ampicillin. The
main characteristics allowing differentiation
among these three species
are summarized in Table
2.
| |
ACKNOWLEDGMENTS |
We are grateful to D. Monnet and M. Fussenegger for translation of
and critical comments on the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: DERBA UPRES EA
1655, Faculté de Médecine RTH Laennec, Laboratoire de
Bactériologie, rue Guillaume Paradin, 69372 Lyon Cedex 08, France. Phone: 33 4 78 77 86 57. Fax: 33 4 78 77 86 58. E-mail:
renaud{at}cimac-res.univ-lyon1.fr.
| |
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Journal of Clinical Microbiology, December 1998, p. 3698-3702, Vol. 36, No. 12
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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