Journal of Clinical Microbiology, May 1998, p. 1430-1432, Vol. 36, No. 5
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification of Corynebacterium
amycolatum and Other Nonlipophilic Fermentative Corynebacteria of
Human Origin
Georges
Wauters,1,*
Bernard
Van
Bosterhaut,2
Michèle
Janssens,1 and
Jan
Verhaegen3
Microbiology Unit, University of Louvain,
1200 Brussels,1
Centre Hospitalier de
Mouscron, 7700 Mouscron,2 and
Department
of Microbiology, University Hospital St-Rafaël, 3000 Leuven,3 Belgium
Received 7 November 1997/Returned for modification 22 January
1998/Accepted 10 February 1998
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ABSTRACT |
Four identification tests, proposed in addition to conventional
methods, were evaluated with 320 fermentative nonlipophilic Corynebacterium strains: growth at 20°C, glucose
fermentation at 42°C, alkalinization of sodium formate, and acid
production from ethylene glycol. These tests were highly discriminant.
Corynebacterium amycolatum displayed a unique profile,
allowing it to be distinguished from similar species, such as C. xerosis, C. striatum, and C. minutissimum.
| |
TEXT |
Nondiphtheric corynebacteria are of
growing importance as opportunistic pathogens, especially in nosocomial
settings (6). Corynebacterium amycolatum is one
of the diphtheroids most often isolated from clinical samples (3,
11, 13). Many conventional tests have variable results with this
species, leading to confusion with other fermentative corynebacteria,
such as C. striatum or C. minutissimum and, more
particularly, C. xerosis (3, 13). Moreover,
C. amycolatum was not included in some commercial
identification systems, resulting in no identification or
misidentification of the strains (1, 13), although it now
appears in the updated API Coryne database 2.0 (5).
Recently, several new nonlipophilic fermentative corynebacteria,
isolated from humans, have been described: C. argentoratense
(12), C. coyleae (4), and C. imitans (2). This may also increase the need to improve
the existing identification schemes by additional discriminant
characteristics.
In this study, we have evaluated some cultural and biochemical
properties not yet applied to the identification of
corynebacteria. These tests are easy to perform in the routine
laboratory and may contribute to the differentiation of nonlipophilic
fermentative corynebacteria.
Bacterial strains.
The following 16 reference strains were
used: C. amycolatum CIP 103452T; C. minutissimum NCTC 10288T and NCTC 10284; C. striatum ATCC 6940T; C. xerosis ATCC
373T, ATCC 7711, and DSM 43607; C. glucuronolyticum DSM 44120T, DMMZ (Department of
Medical Microbiology, Zürich) 845, and DMMZ 987; C. argentoratense CIP 104296T; C. coyleae DSM
44184T and DSM 44185; C. imitans DSM
44264T; and C. diphtheriae DSM
44123T and DSM 43988.
Three hundred four isolates of nonlipophilic fermentative
corynebacteria of human origin were included in the study. They were
collected from various clinical samples by several laboratories. Strains were identified according to existing identification schemes by
the methods described by other authors (6, 7, 12, 13). Production of propionic acid from glucose was detected in glucose broth
by gas-liquid chromatography in all strains as previously described
(13).
Identification tests proposed in this study. (i) Growth at
20°C.
A suspension (±2 McFarland standard) was made from a 24-h
blood agar culture. One loopful was evenly streaked onto the surface of
half of a blood agar plate and incubated at 20 ± 1°C. Obvious macroscopic growth was recorded after 1, 2, and 3 days.
(ii) Glucose fermentation at 42°C.
The same suspension
described for growth at 20°C was used for glucose fermentation at
42°C. Two drops were inoculated into 4 ml of phenol red broth base
(Difco, Detroit, Mich.) supplemented with 1% (wt/vol) glucose and
0.1% (vol/vol) Tween 80. Tubes were incubated in a water bath at
42°C (±0.1°C) for 3 days. A yellow color shift was recorded as a
positive fermentation test.
(iii) Alkalinization of a sodium formate solution.
Strains
were cultured for 24 h on brain heart agar, heart infusion agar,
or brucella agar. No tryptic soy agar or blood-supplemented media
should be used. A heavy suspension (4 to 6 McFarland standard) was made
in 0.5 ml of distilled water distributed in small tubes, and 0.5 ml of
the following solution was added: sodium formate, 5 g; 0.5%
cresol red solution, 1 ml; 0.001 M phosphate buffer (pH 6), 100 ml.
After overnight incubation at 37°C, positive reactions were
characterized by a purple color shift. Negative reactions were yellow.
Additional readings were made after 2 and 3 days.
(iv) Acid production from ethylene glycol in a low-peptone-content
medium.
Acid production was measured with a low-peptone-medium
consisting of casitone (Difco) (1 g), ethylene glycol (20 ml), NaCl (5 g), agar (17 g), phenol red (0.025 g), and distilled water (1 liter).
No buffer was added, and the pH was adjusted to ±7.2. After melting,
the medium was distributed in 5-ml amounts in tubes and distributed to
slants after sterilization. The slants were heavily inoculated with
bacteria from a 24-h blood agar plate and incubated for 2 days. The
medium turned yellow, usually within 24 h, when acid was produced
from ethylene glycol.
Genetic studies.
Strain K132 was submitted to S. Pradella,
Deutsche Sammlung von Mikroorganismen und Zelkulturen (DSMZ),
Braunschweig, Germany, for genomic analysis. Approximately 95% of the
16S rRNA gene sequences of this strain were determined by direct
sequencing of PCR-amplified 16S ribosomal DNA (rDNA). Genomic DNA
extraction, PCR-mediated amplification of the 16S rDNA, and
purification of the PCR products were carried out as described
previously (9). Purified PCR products were sequenced with
the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit
(Applied Biosystems, Weiterstadt, Germany) as directed in the
manufacturer's protocol. Sequence reaction mixtures were
electrophoresed with the Applied Biosystems 373A DNA Sequencer. The
resulting sequence data from strain K132 were put into the alignment
editor ae2 (8), aligned manually, and compared with the 16S
rDNA sequence of C. xerosis ATCC 373T (AC
X81914).
Results and discussion.
Identification by conventional methods
and detection of propionic acid production allowed us to assign the 304 clinical strains to the following species: 210 were C. amycolatum, 34 were C. minutissimum, 29 were C. striatum, 15 were C. diphtheriae, 14 were C. glucuronolyticum, 1 was C. xerosis, and 1 was C. imitans.
As expected, C. amycolatum strains were variable in
fermentation tests of sucrose, maltose, and galactose and in nitrate
reduction or urease. Three strains exhibited 
-glucosidase activity.
All of the strains formed dry colonies and produced propionic acid from
glucose.
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