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Journal of Clinical Microbiology, September 2001, p. 3104-3109, Vol. 39, No. 9
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3104-3109.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Identification of Strains of
Alcaligenes and Agrobacterium by a
Polyphasic Approach
Dominique
Clermont,*
Christine
Harmant, and
Chantal
Bizet
Collection de l'Institut Pasteur, 75724 Paris Cédex 15, France
Received 15 February 2001/Returned for modification 23 April
2001/Accepted 8 July 2001
 |
ABSTRACT |
The number of stable discriminant biochemical characters is limited
in the genera Alcaligenes and
Agrobacterium, whose species are consequently difficult
to distinguish from one another by conventional tests. Moreover,
genomic studies have recently drastically modified the nomenclature of
these genera; for example, Alcaligenes xylosoxidans was
transferred to the genus Achromobacter in 1998. Twenty-five strains of Achromobacter xylosoxidans, three
strains of an Agrobacterium sp., five strains of an
Alcaligenes sp., and four unnamed strains belonging to
the Centers for Disease Control and Prevention group IVc-2 were
examined. These strains were characterized by conventional tests,
including biochemical tests. The assimilation of 99 carbohydrates,
organic acids, and amino acids was studied by using Biotype-100 strips,
and rRNA gene restriction patterns were obtained with the automated
Riboprinter microbial characterization system after cleavage of total
DNA with EcoRI or PstI restriction endonuclease. This polyphasic approach allowed the two subspecies of
A. xylosoxidans to be clearly separated. Relationships
between five strains and the Ralstonia paucula type
strain were demonstrated. Likewise, three strains were found to be
related to the Ochrobactrum anthropi type strain. We
showed that substrate assimilation tests and automated ribotyping
provide a simple, rapid, and reliable means of identifying A.
xylosoxidans subspecies and that these two methods can be used
as alternative methods to characterize unidentified strains rapidly
when discriminant biochemical characters are missing.
| |
INTRODUCTION |
The taxonomic position of the
genus Alcaligenes has been changing for a few years as a
result of genomic studies. Alcaligenes species have been
transferred to the genera Carbophilus (14), Halomonas (9), Ralstonia (18,
20), and Variovorax (19). Alcaligenes xylosoxidans, Alcaligenes ruhlandii,
and Alcaligenes piechaudii were recently reassigned to the
genus Achromobacter (21). The subspecies
Achromobacter xylosoxidans subsp. denitrificans has been proposed and the subspecies Achromobacter
xylosoxidans subsp. xylosoxidans was automatically
created. Likewise, some Agrobacterium species now belong to
the genera Ruegeria and Stappia (17).
A. xylosoxidans was found in aqueous environmental sources
and isolated from a wide range of clinical samples. This organism is
recognized as an opportunistic pathogen responsible for serious infections (8, 10). Medical equipment and solutions have been found to be contaminated with this organism (8).
Due to the limited number of stable discriminating characteristics,
many Alcaligenes and Agrobacterium species remain
difficult to distinguish from one another by conventional tests.
Ribotyping was proposed as a taxonomic tool a few years ago and was
shown to differentiate genera and species (4, 11, 13, 15,
16). More recently, a phenotypic approach based on an auxanogram
using the Biotype-100 identification system revealed the taxonomic
diversity of the pseudomonads (12) and also successfully
distinguished Rhodococcus and Gordonia strains
(2).
Twenty-five strains of A. xylosoxidans subsp.
xylosoxidans or subsp. denitrificans, five
Alcaligenes strains, three Agrobacterium strains,
and four unnamed bacteria belonging to the Centers for Disease Control
and Prevention (CDC) group IVc-2 from the Collection de l'Institut
Pasteur (CIP) were identified to the species or subspecies level by a
polyphasic approach based on conventional tests, auxanogram, and
automated ribotyping.
| |
MATERIALS AND METHODS |
Bacterial strains.
Twenty-five strains of A. xylosoxidans, three strains of Agrobacterium, five
strains of Alcaligenes, and four unnamed strains belonging
to CDC group IVc-2 were studied. All the strains belonged to the CIP
(Table 1).
Conventional identification.
All strains were examined by
the conventional tests described by Chester and Cooper
(7).
Identification with Biotype-100 strips.
The assimilation of
99 carbohydrates, organic acids, and amino acids was studied by using
Biotype-100 strips (BioMérieux, La Balme-les Grottes,
France). Growth after 1 and 4 days of incubation was compared with that
of the control without carbon source. The Recognizer, Adanson,
and Dendrograf programs of the Taxotron package were used according to
the user's manual for numerical analysis of Biotype-100 data. The
distance coefficient selected was the complement of the Jaccard
coefficient, and clustering was done by the unweighted pair group
method of averages.
Identification with ribotyping method.
Ribotyping was
carried out using the Riboprinter microbial characterization system
(Qualicon, Inc., Wilmington, Del.). Colonies were picked from solid
medium, suspended in sample buffer, and heat treated. The lysing agent
was added, and the samples were transferred to the Riboprinter system.
Restriction endonuclease digestion, gel separation, transfer, and
hybridization with a chemiluminescence-labeled DNA probe containing the
rRNA operon from Escherichia coli were carried out by the
automated instrument in 8 h.
Gel images were exported in TIFF and analyzed with the RestrictoScan,
Restrictotyper, Adanson, and Dendrograf programs of
the Taxotron
package. The cubic Spline algorithm was used to calculate
fragment
sizes. A fixed fragment size tolerance value of 4% was
chosen.
Antibiotic susceptibility.
Antibiotic susceptibility of the
strains was tested by the agar diffusion method (5). The
susceptibilities were determined according to the guidelines of the
Comité de l'Antibiogramme de la Société
Française de Microbiologie (1).
| |
RESULTS |
Conventional identification.
All the strains studied were
gram-negative rods, motile, oxidase and catalase positive, and strictly
aerobic. All the strains were negative for production of gelatinase,
caseinase, 
-galactosidase, DNase, and arginine-dihydrolase. Lysine
and ornithine were also not decarboxylated.
The
A. xylosoxidans strains were characterized by their
capacity to grow anaerobically with nitrate as the electron acceptor
and by the absence of enzymatic activities as well as by the lack
of
production of H
2S and hydrolysis of esculin and
tributyrin.
Characteristics differentiating the 25 strains of
A. xylosoxidans are shown in Table
2.
Xylose oxidative degradation was the only
positive test for the 20
A. xylosoxidans subsp.
xylosoxidans strains.
This
test was negative for the five
A. xylosoxidans subsp.
denitrificans strains.
For the 12 remaining strains, the results obtained for conventional
tests are shown in Table
3.
Alcaligenes strains showed
great variability in their
conventional tests, whereas
Agrobacterium strains had a lot
of common traits. The unnamed bacteria all gave
identical responses to
the tests carried out.
Biotype data.
The 37 strains studied could all use a wide
range of organic compounds as their sole energy and growth sources. The
use of L-tartrate, trans-aconitate,
D-gluconate, and caprate as carbon sources
allowed us to differentiate the two subspecies of A. xylosoxidans. Whereas A. xylosoxidans subsp.
xylosoxidans was able to grow on trans-aconitate,
D-gluconate, and caprate, A. xylosoxidans subsp. denitrificans was not. In contrast,
the A. xylosoxidans subsp. denitrificans used
L-tartrate while A. xylosoxidans
subsp. xylosoxidans did not. The phenogram in Fig.
1 shows the relationships, in terms of
carbon source utilization, between these strains and the A. xylosoxidans subsp. xylosoxidans, A. xylosoxidans subsp. denitrificans, Ralstonia
paucula, and Ochrobactrum anthropi type strains.
Examination of the phenogram gave clear groupings and allowed us to
define four phenogroups (A, B, C, and D) for the 41 strains tested.
Phenogroup A was composed of A. xylosoxidans subsp.
xylosoxidans strains. Two strains, CIP 104044 and CIP
104045, were more closely related to the type strain of A. xylosoxidans subsp. xylosoxidans than the others.
Phenogroup B included the A. xylosoxidans subsp.
denitrificans strains. The five strains of
Alcaligenes and the four unnamed bacteria fell into
phenogroup C, as did the R. paucula type strain. Phenogroup
D contained the three Agrobacterium strains studied and the
O. anthropi type strain.
Ribotyping data.
Ribotyping of the 41 strains listed in Table
1 was carried out by an automated system. The EcoRI and
PstI enzymes always generated an appropriate number of
restriction fragments to allow a comparative analysis to be made. A
schematic representation of the banding patterns of all the strains and
the deduced dendrograms are shown in Fig.
2 and 3.
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|
FIG. 2.
Dendrogram based on the EcoRI ribotyping
patterns of the 41 strains. A. xyl. xyl., A.
xylosoxidans subsp. xylosidans; A. xyl.
denit., A. xylosoxidans subsp.
denitrificans.
|
|
The dendrogram obtained from
EcoRI patterns revealed four
ribogroups (Fig.
2). Ribogroup I included all the strains of
A. xylosoxidans subsp.
xylosoxidans. It was homogeneous
and composed
mostly of strains with a pattern identical to that of the
species
type strain. Most of the differences observed were within the
smallest restriction fragments. Ribogroup III was composed of
strains
with a pattern similar or identical to that of the
A. xylosoxidans subsp.
denitrificans type strain.
Ribogroup II, including
the
Alcaligenes strains and the four
unnamed bacteria, could be
subdivided into three clusters. In the first
cluster the profiles
observed for each strain were very similar to that
of the
R. paucula type strain and differed significantly
from those of the two other
clusters. Ribogroup IV was subdivided into
two clusters, one containing
two
Agrobacterium strains and
the other including the
O. anthropi type strain and
Agrobacterium sp. strain CIP 102731. These last
two strains
had identical ribotyping patterns. It is interesting
that resistance to
ticarcillin, piperacillin, cephalothin, cefotaxime,
ceftazidime,
kanamycin, and erythromycin and susceptibility to
gentamicin and
nalidixic acid were observed for
Agrobacterium strains and
for the
O. anthropi type
strain.
To verify whether
EcoRI groupings were of taxonomic
significance for the unidentified strains, they were ribotyped after
digestion
with
PstI. The resulting dendrogram (Fig.
3) shows
that
Alcaligenes and
Agrobacterium strains were
separated into two groups. The
R. paucula type strain was
found in one of these groups, and the
O. anthropi type
strain was in the other. A significant distance
between three of the
Alcaligenes strains and
R. paucula type strain
was observed. This result was in agreement with that obtained
with
EcoRI for two of the strains. The third strain, CIP 101080,
appeared to be more distant from
R. paucula when cut with
PstI
than with
EcoRI. Conversely, CIP 100008 appeared to be closer
to
R. paucula when cut with
PstI than with
EcoRI. The
EcoRI and
PstI results both gave the same groupings for the
Agrobacterium strains.
| |
DISCUSSION |
We carried out a polyphasic taxonomic study to identify 37 strains. The strains were characterized by use of an auxanogram and
ribotyping. The results were analyzed by computer and compared to the
results given by conventional tests.
The oxidative degradation of xylose was the only biochemical
characteristic found to differentiate the two A. xylosoxidans subspecies. Due to the lack of discriminating
biochemical features, reliable tests were needed to distinguish the two
subspecies. Our investigation demonstrated that although ribotyping
cannot delineate the Staphylococcus subspecies
(6), it can clearly discriminate the two A. xylosoxidans subspecies. The ribogroups obtained were homogeneous
with respect to current nomenclature, and it is noteworthy that most
A. xylosoxidans subspecies strains displayed identical
ribotyping patterns. Moreover, ribogroups were consistent with
phenogroups. However, the biotype data suggested that the two
subspecies were more closely related than the ribotyping data.
Without consideration of the biochemical behavior, ribotyping and
substrate assimilation tests grouped one strain of
Alcaligenes, CIP 102485, and the four unnamed strains
(CIP 104521, CIP 104522, CIP 104523, and CIP 104524) to R. paucula. Likewise, three strains of Agrobacterium, CIP
102460, CIP 102731, and CIP 102250, were related to the O. anthropi type strain.
It was not surprising that the unnamed bacteria were found to be
related to R. paucula, because they belong to the same CDC group as CIP 105943T (18). The
observation that the ribotypes of unidentified strains fall into the
same cluster as the R. paucula or O. anthropi
type strains in both ribotyping assays strongly suggests that they belong to the corresponding species. Using two endonucleases instead of
one reduces the probability of two unrelated strains having similar
patterns. CIP 102460, CIP 102731, and CIP 102250 also had drug
resistance profiles similar to that of O. anthropi, which also suggested that these strains belong to this species
(3).
Although the ribotype and biotype groupings corresponded, the
respective distribution of the strains within each group was slightly
different. This explains why it was difficult to classify CIP 101080 and CIP 100008. Furthermore, the comparison with other type strains of
neighboring species, like Ralstonia pickettii, Ralstonia gilardii, Ralstonia eutropha, and
Ralstonia solanacearum, did not provide the best grouping
for CIP 100998 and CIP 10007 (data not shown). Further analysis is
required to resolve the taxonomic relationship of CIP 101080, CIP
100008, CIP 100998, and CIP 10007. Methods that may be used include 16S
rRNA sequencing, followed by the comparison of the sequences obtained
to those found in the databases.
This study emphasized the need for performant bacterial strain
identification systems and the creation of databases which allow the
strains identified by each system to be compared. The methods used in
this study constitute a powerful tool for the rapid, simple, and
reliable identification of strains which are difficult to separate
using conventional tests. These methods cannot replace quantitative
DNA-DNA hybridization studies for the estimation of relationships
between the strains; however, they allow the rapid screening of
unidentified strains for comparison with the type strains of species
within the same genus.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Collection de
l'Institut Pasteur, BP 52, 25 Rue du Docteur Roux, 75724 Paris
Cédex 15, France. Phone: 01 40 61 31 04. Fax: 01 40 61 30 07. E-mail: dclermon{at}pasteur.fr.
| |
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Journal of Clinical Microbiology, September 2001, p. 3104-3109, Vol. 39, No. 9
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3104-3109.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
