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Previous Article | Next Article 
Journal of Clinical Microbiology, October 1999, p. 3194-3197, Vol. 37, No. 10
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Diverse Restriction Fragment Length Polymorphism
Patterns of the PCR-Amplified 16S rRNA Genes in Aeromonas
veronii Strains and Possible Misidentification of
Aeromonas Species
Joerg
Graf*
Institute for Medical Microbiology,
University of Berne, CH-3010 Berne, Switzerland
Received 20 April 1999/Returned for modification 4 June
1999/Accepted 9 July 1999
 |
ABSTRACT |
Restriction fragment length polymorphism analysis after PCR
amplification (RFLP-PCR) of the 16S rRNA gene has been previously proposed as a rapid method to identify Aeromonas species.
In the present study, the precision of RFLP-PCR was evaluated with 62 Aeromonas reference strains. The analysis revealed that
Aeromonas veronii biovar sobria strains produce various
patterns, possibly leading to its misidentification as an environmental
species. For most other Aeromonas species little variation
was noted. This study supports the usefulness of RFLP-PCR analysis to
separate three clinically important species but also reveals possible
misidentifications that necessitate further biochemical tests to
validate the preliminary identification.
| |
INTRODUCTION |
For the identification of bacteria,
the speed and precision of molecular approaches are attractive to many
investigators, and such approaches can be used as either complements or
alternatives to biochemical identification (5, 8, 23).
Because the biochemical identification of Aeromonas species
can be time-consuming and difficult (1, 2), there has been
an interest in finding molecular methods. Promising approaches with
species-specific PCR primers have been reported (7, 9, 19).
Recently, a more powerful method has been published that relies on the
use of a single pair of primers to amplify the DNA encoding a variable region of the 16S rRNA gene (4). The amplified DNA is
subsequently subjected to restriction fragment length polymorphism
(RFLP) analysis. Using this RFLP-PCR approach, the authors were able to
separate most Aeromonas species and to identify unknown
isolates (4). The validity of the method needed to be
evaluated with multiple reference strains (8), because only
one reference strain was tested for each species (4) and
very small differences have been reported in the sequence of the
amplified 16S rRNA gene (20).
| |
MATERIALS AND METHODS |
Bacterial strains and growth conditions.
The strains used in
this study were identified to the species level by DNA-DNA
hybridization by other groups (Table 1).
The Aeromonas strains were grown on sheep blood agar at
30°C (11).
RFLP-PCR conditions.
The PCR for the RFLP-PCR was done as
described previously (11). The sequence of the forward
primer was 5'-TCA TGG CTC AGA TTG AAC GCT-3', and the reverse primer
was 5'-CGG GGC TTT CAC ATC TAA CTT ATC-3' (MWG-Biotech, Ebersberg,
Germany). After overnight growth on a blood agar plate, a single colony
was resuspended in 20 µl of saline solution and the sample was placed
into a boiling water bath for 10 min. Ten volumes of TE (Tris-EDTA, pH
7.6 [24]) was added, and the solution containing the
DNA was stored at 
20°C until use. One microliter of the DNA
solution was added to the PCR mixture containing 1.5 mM
MgCl2 (Boehringer Mannheim, Mannheim, Germany). The
amplification conditions were as follows: (i) 1 cycle consisting of
94°C for 5 min; (ii) 30 cycles consisting of 94°C for 1 min, 57°C
for 1 min, and 72°C for 1 min; and (iii) 1 cycle consisting of 94°C
for 1 min, 57°C for 1 min, and 72°C for 2 min. For the restriction
digests, 3.5 µl of the amplified sample was digested with 10 U of
either AluI, CfoI, or MnlI (Boehringer Mannheim and New England Biolabs, Beverly, Mass.). The digested samples
were analyzed by agarose gel electrophoresis (3% ResoPhor [Eurobio,
Les Ullis Cedex B, France]; 10 V/cm).
Biochemical identification.
Representative strains from
different RFLP patterns were selected to verify their identities by
biochemical testing. The identification scheme was the same as we used
in a previous study (11). It is a biochemical test scheme
that combines the results from several studies (1, 10, 12, 14,
16). Most of the test results were obtained with the test strips
API 20 NE and API 20 E (BioMérieux, Marcy l'Etoile, France)
according to the manufacturer's instructions. For Aeromonas
encheleia, the API 20 E tests were done at 28°C. Gas production
from glucose was determined by using triple sugar iron agar. Resistance
to cephalothin and to ampicillin (30 and 10 µg per Sensi-Disc,
respectively; Becton Dickinson Europe, Meylan Cedex, France) was
determined on blood agar plates at 30°C. Lactose fermentation was
tested on MacConkey lactose agar (Oxoid, Columbia, Md.) after 18 h
at 30°C.
| |
RESULTS AND DISCUSSION |
Various RFLP-PCR patterns for Aeromonas veronii biovar
sobria.
The ability to accurately identify Aeromonas
species by RFLP-PCR analysis of the 16S rRNA gene was evaluated by
comparing the RFLP patterns of 62 reference strains (Table
2 and Fig.
1). The digestion of the PCR product with
the restriction endonuclease AluI revealed unexpected
variation for A. veronii. A previous study reported no
differences for the sequence of the 16S rRNA gene within A. veronii, and the same DNA sequence was reported for the two known
biovars, A. veronii biovar sobria and A. veronii biovar veronii (20). While we observed the expected RFLP
pattern (F) for both biovars, a second pattern (B/d) was detected for three A. veronii biovar sobria strains (CDC 0437-84, A 155, and A 916). This RFLP pattern corresponded to the pattern observed for
Aeromonas media and Aeromonas allosaccharophila,
possibly leading to a misidentification of A. veronii. Both
of these species are considered to be environmental species, not human
pathogens (15, 21).
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TABLE 2.
Number of reference strains exhibiting the indicated RFLP
patterns obtained from restriction digests of the amplified
variable region of the 16S rRNA gene
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FIG. 1.
RFLP patterns of the PCR-amplified gene encoding 16S
rRNA from Aeromonas reference strains. Seven different RFLP
patterns (A to G) were observed for AluI restriction
digests. Three patterns (a to c) were observed for CfoI
digests, and two patterns (d and e) were seen for MnlI.
These patterns correspond to the patterns indicated in Table 2. The
molecular weight standard (Std) was pBR322 DNA digested with
MspI (New England Biolabs). The gels were photographed with
Bio-print (version 6.21; Vilber Lourmat, Marne la Vallée,
France), exported into Adobe Photoshop, and labeled in Macromedia
Freehand 7.0.
|
|
Using different approaches, other studies have reported tremendous
heterogeneity within A. veronii biovar sobria. The classic study by Popoff et al. (22) revealed that A. veronii biovar sobria was comprised of two distinct DNA-DNA
hybridization groups (HG 8X and 8Y). Using a numerical classification
approach, Kämpfer and Altwegg (18) described four
biochemically distinct and very diverse clusters (clusters 1, 5, 6, and
8) of A. veronii strains. Similarly, Carnahan and Joseph
(6) found that the type strain for HG 8X, CDC 0437-84 with
RFLP pattern B/d, greatly differed phenotypically from other A. veronii biovar sobria strains. Multilocus enzyme analysis revealed
four distinct phenons (groups of similar strains) for A. veronii biovar sobria (phenons 1, 3, 4, and 13), and the strains
with RFLP pattern B/d belonged to phenon 3 (3). Studies
analyzing the similarity of the genome from Aeromonas strains by comparing amplified fragment length polymorphism
demonstrated that some A. veronii biovar sobria strains were
more closely related to A. veronii biovar veronii while
others (A 155 and A 916; RFLP pattern B/d) grouped with A. allosaccharophila (RFLP pattern Bld [13]).
Finally, a mouse virulence model demonstrated a large variation in
pathogenicity for A. veronii biovar sobria (17). Three of the A. veronii biovar sobria strains tested were
included in this study. The least virulent A. veronii biovar
sobria strain had pattern B/d (50% lethal dose, 9.0), while the other
two strains had pattern F and were much more virulent (50% lethal
dose, 6.6 and 7.2 [17]). When different criteria were
analyzed, all of these studies revealed large variation within the
species A. veronii, much larger than that observed for most
other Aeromonas species. In several of these
characteristics, ranging from biochemical tests to virulence, the
A. veronii biovar sobria strains with RFLP pattern B/d
differed from the strains with RFLP pattern F. These results suggest
that variation in the 16S rRNA gene of A. veronii biovar
sobria may reflect, and help to clarify, some of the phenotypic
variation observed in this species.
RFLP-PCR patterns of other aeromonads.
In contrast to the
different 16S rRNA patterns observed for A. veronii, most
other Aeromonas species (10 species) exhibited a single RFLP
pattern, reflecting the relatively conserved nature of the 16S rRNA
gene in Aeromonas. In addition to A. veronii, only A. media (two of eight strains tested) and A. encheleia (two of six) isolates exhibited alternative RFLP
patterns (Table 2). For A. media, these differences also
reflected the phenotypic differences detected by Altwegg et al.
(3), further suggesting that the variability in the sequence
of the 16S rRNA gene may correlate with phenotypic differences.
Biochemical verification of species identification.
The
additional RFLP patterns observed within a species could be due to
actual variation of the 16S rRNA gene sequence or to false species
identification. Thus, we attempted to verify the identities of strains
with different RFLP patterns by biochemical tests. Of the five A. veronii biovar sobria strains verified, the test results for the
two strains with RFLP patterns D and F were identical to those reported
for A. veronii biovar sobria (1, 12, 14). For one
strain (CDC 0437-84; RFLP pattern B/d) which had been previously
reported to differ phenotypically from other Aeromonas
veronii biovar sobria strains (e.g., negative for the
Voges-Proskauer test [6]), we obtained the same test results that had been reported previously, thus confirming its identity. A second strain, also with RFLP pattern B/d, differed in the
biochemical tests from CDC 0437-84 only by a very weak esculin
reaction. In a previous study, we had isolated A. veronii biovar sobria strains from the medicinal leech, Hirudo
medicinalis, that also had a positive esculin reaction
(11). The fifth A. veronii biovar sobria strain,
with RFLP pattern E/a, had unusual test results for A. veronii biovar sobria. It was esculin positive, Voges-Proskauer
negative, and resistant to cephalothin. These results are in agreement
with an identification as Aeromonas trota (RFLP pattern C).
However, the strain was resistant to ampicillin, whereas A. trota is characteristically sensitive. The biochemical identification of this strain is uncertain, but the strain may be an
unusual A. veronii. Thus four of the five strains tested are
clearly A. veronii biovar sobria strains, suggesting that the variation in the RFLP-PCR patterns observed is due to differences in the sequences of the 16S rRNA genes.
The biochemical tests confirmed the identification of the three
A. media strains (patterns A, B, and C [12,
16]). The two A. encheleia strains (patterns A and
B) had test results identical to those reported previously except that
they were negative for acid production from sucrose (14,
21). However in the original description one of the four strains
examined was also negative for this test (21), suggesting
that these strains are A. encheleia. One strain,
Aeromonas jandaei (RFLP pattern A/a), was excluded from the
study because it was biochemically similar to Aeromonas hydrophila (RFLP pattern A/a). The 10 strains that were verified included 7 strains with unexpected RFLP patterns. These results suggest
that the variation in RFLP patterns observed in our study are unlikely
to be due to strain switching or incorrect identification.
RFLP-PCR analysis of aeromonads.
The additional RFLP patterns
within species and the lack of detectable differences between other
species clearly limit the use of RFLP-PCR for species identification in
Aeromonas. However, the three most frequently encountered
clinical specimens (15), A. hydrophila (pattern
A), Aeromonas caviae (C), and A. veronii biovar
sobria (B and F) could be separated in all cases by using only the
restriction enzyme AluI. Thus, RFLP-PCR of the 16S rRNA gene
allows a rapid, presumptive species identification. If one excludes the
RFLP patterns from infrequent isolates (single strains differing from
the usual pattern of a species), one subsequent digest with a single
additional restriction enzyme allows unambiguous species identification
for seven species (Table 2). An additional four species could be
grouped into pairs according to RFLP patterns (Aeromonas
bestiarum-Aeromonas salmonicida and A. caviae-A.
trota). The initial study by Borell et al. used a similar RFLP-PCR
identification scheme to identify numerous clinical isolates
(4). These results suggest that RFLP-PCR of the 16S rRNA
gene can be used as a tool to aid in initial Aeromonas
species identification. However, verification of species identification
with biochemical tests is still appropriate for clinical diagnosis in
light of the differences reported in our study.
The results presented in this study demonstrate variation in the 16S
rRNA gene of A. veronii biovar sobria and reveal possible false identifications by RFLP-PCR analysis of the 16S rRNA. While molecular approaches provide fast and apparently straightforward results, problems can arise if not enough reference strains are used in
establishing a broad database to validate the methodology (8). Future studies, such as sequencing the 16S rRNA gene
from strains belonging to the different phenons, may help to explain the phenotypic variation observed in A. veronii and allow
for the separation of biovars that do not present a clinical problem.
| |
ACKNOWLEDGMENTS |
I thank K. Schopfer and M. Täuber for their encouragement
to pursue this study. I am especially thankful to K. Mühlemann for helpful discussion and A. Carnahan and E. S. Mirkin for
commenting on the manuscript. I am grateful to M. Altwegg, J. M. Janda, and R. Powell for sending Aeromonas reference strains
and R. Troller for excellent technical assistance with the biochemical identification.
I thank the Institute for Medical Microbiology of the University of
Berne for financial support.
| |
FOOTNOTES |
*
Mailing address: Institute for Medical Microbiology,
University of Berne, Friedbühlstr. 51, CH-3010 Berne,
Switzerland. Phone: 41-31-632-3568. Fax: 41-31-632-3550. E-mail:
jgraf{at}imm.unibe.ch.
| |
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Journal of Clinical Microbiology, October 1999, p. 3194-3197, Vol. 37, No. 10
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
