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Journal of Clinical Microbiology, May 2000, p. 2023-2025, Vol. 38, No. 5
0095-1137/00/$04.00+0
LETTERS TO THE EDITOR
Use of Restriction Fragment Length Polymorphism of the
PCR-Amplified 16S rRNA Gene for the Identification of
Aeromonas spp.
 |
LETTER |
In the October 1999 issue of the Journal of Clinical
Microbiology, Graf (4) presented a paper the results of
which demonstrated variations in the restriction fragment length
polymorphism (RFLP) patterns of the PCR-amplified 16S rRNA gene of
Aeromonas veronii biovar sobria and possible false
identifications resulting from this technique. The author pointed out
that although 16S rDNA-RFLP had been proposed as a rapid method of
identifying Aeromonas species (1), its precision
needed to be evaluated with more reference strains, because only one
reference strain had been tested for each species. In his work, Graf
(4) used 62 strains from several collections to verify our
protocol, but an RFLP method that was different from the one previously
proposed by Borrell et al. (1) was used. Essential
differences between the methods were as follows: (i) only a part of the
16S rRNA gene was amplified by Graf (600 bp at the 5' end) instead of
the complete gene as we proposed and (ii) the enzymes employed
(AluI, CfoI, and MnlI) to obtain the
species-specific patterns were not the ones originally proposed. These
important differences are not mentioned at all in Graf's paper
(4). The simultaneous application of AluI and
MobI allowed the separation of 10 species of
Aeromonas, as stressed by Borrell et al. (1). The
endonucleases in our study were the ones that, after computer analysis,
targeted the species-specific regions within the entire 16S rRNA gene
(8). The method was evaluated by using the type strain of
each species, along with 76 previously biochemically identified strains
(1), and it has also been used to characterize 55 strains of
A. veronii, always producing unequivocal patterns (M. J. Figueras, A. J. Martinez-Murcia, N. Borrell, and J. Guarro,
Abstr. 99th Gen. Meet. Am. Soc. Microbiol., 1999, abstr. C-401, p. 187, 1999). In Graf's paper (4), the criteria for selecting the
enzymes are not mentioned but do not appear to be based on any
previously computerized analysis of the 16S rRNA gene sequences of the
type strains. This is evident from the nondiscriminatory patterns
obtained for some species. For instance, strains ATCC 35941 and LMG
13076, which belong to Aeromonas sp. HG11, showed RFLP
patterns identical to that of Aeromonas encheleia
(1). However, there are eight nucleotide differences in the
16S ribosomal DNA (rDNA) sequence (9), and although
Aeromonas sp. HG11 and A. encheleia are
considered the same species by some authors (6), the
proposal has not been formally validated since data contradicts the
original description that is based on phenotype and DNA-DNA pairing
(3). Theoretical computer analysis of the type strains also
prevents misinterpretation, such as that caused by undigested fragments
at the laboratory, as shown in the 325-bp band in lanes B and G of Fig.
1 in Graf's work (4), which must not appear.
As already noted in our paper (1), 16S-rDNA RFLP patterns
different from those previously described may be expected if the
digested sequence belongs to a new Aeromonas species or if the restriction sites in known species are affected by intraspecific nucleotide diversity. To date, the RFLP method we proposed has been
successfully tested with more than 200 strains, including numerous
reference strains, and when a different pattern has been encountered
(unpublished results), it has corresponded to the newly described
species Aeromonas popoffii (7). A recent study found variations in five nucleotide positions after the 16S rRNA gene
of 12 A. popoffii strains was sequenced, but despite these variations, the existence of unique primary structures in the gene was
recognized as useful for its identification (2).
The intraspecific heterogeneity reported by Graf may be due to the use
of some reference strains that in previous papers showed contradictory
results. An example of this is ATCC 43946, the strain that was wrongly
included as A. encheleia in Graf's list. While DNA-DNA
hybridizations of this strain show that it is closely related to
Aeromonas schubertii, it belongs in fact to the
Aeromonas group 501 (5), which has 30 nucleotides
that are different in the 16S rRNA gene sequence from that of
A. encheleia (9). Graf interprets the
different patterns as a case of intraspecific diversity (4).
It is also worth mentioning that Graf misinterprets the variations in
the biochemical behavior encountered in some A. veronii biovar sobria strains as being due to differences in the 16S rRNA gene
(4), because phenotypic responses are never under the control of this gene.
In conclusion, Graf's paper (4) should be considered a
modification of the original protocol (1) and in no way
demonstrates its precision. We strongly believe that for a method to be
validated and its precision to be demonstrated with more reference
strains, as was Graf's aim (4), it must be followed in
detail before any conclusions can be made. Finally, it would be a pity
if confusing results obtained by Graf discouraged other researchers
from applying the protocol as originally described (1).
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FOOTNOTES |
*
Phone: 34-977759321
Fax: 34-977759322
E-mail: mjfs{at}fmcs.urv.es
| |
REFERENCES |
| 1.
|
Borrell, N.,
S. G. Acinas,
M. J. Figueras, and A. Martínez-Murcia.
1997.
Identification of Aeromonas clinical isolates by restriction fragment length polymorphism of PCR-amplified 16S rRNA genes.
J. Clin. Microbiol.
35:1671-1674[Abstract].
|
| 2.
|
Demarta, A.,
M. Tonolla,
A.-P. Caminada,
N. Ruggeri, and R. Peduzzi.
1999.
Signature region within the 16S rDNA sequences of Aeromonas popoffii.
FEMS Microbiol. Lett.
172:239-246[CrossRef][Medline].
|
| 3.
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Esteve, C.,
M. C. Gutiérrez, and A. Ventosa.
1995.
Aeromonas encheleia sp. nov. isolated from European eels.
Int. Syst. Bacteriol.
45:462-466[Abstract/Free Full Text].
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| 4.
|
Graf, J.
1999.
Diverse restriction fragment length polymorphism patterns of the PCR-amplified 16S rRNA genes in Aeromonas veronii strains and possible misidentification of Aeromonas species.
J. Clin. Microbiol.
37:3194-3197[Abstract/Free Full Text].
|
| 5.
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Hickman-Brenner, F. W.,
G. R. Fanning,
M. J. Arduino,
D. J. Brenner, and J. J. Farmer, III.
1988.
Aeromonas schubertii, a new mannitol-negative species found in human clinical specimens.
J. Clin. Microbiol.
26:1561-1564[Abstract/Free Full Text].
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| 6.
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Huys, G.,
P. Kampfer,
M. Altwegg,
R. Coopman,
P. Janssen,
M. Gillis, and K. Kersters.
1997.
Inclusion of Aeromonas DNA hybridization group 11 in Aeromonas encheleia and extended descriptions of the species Aeromonas eucrenophila and A. encheleia.
Int. J. Syst. Bacteriol.
47:1157-1164[Abstract/Free Full Text].
|
| 7.
|
Huys, G.,
P. Kampfer,
M. Altwegg,
I. Kersters,
A. Lamb,
R. Coopman,
J. Luthy-Hottenstein,
M. Vancanneyt,
P. Janssen, and K. Kersters.
1997.
Aeromonas popoffii sp. nov., a mesophilic bacterium isolated from drinking water production plants and reservoirs.
Int. J. Syst. Bacteriol.
47:1165-1171[Abstract/Free Full Text].
|
| 8.
|
Martínez-Murcia, A.,
S. Benlloch, and D. Collins.
1992.
Phylogenetic interrelationships of members of the genera Aeromonas and Plesiomonas as determined by 16s ribosomal DNA sequencing: lack of congruence with results of DNA-DNA hybridizations.
Int. J. Syst. Bacteriol.
42:412-421[Abstract/Free Full Text].
|
| 9.
|
Martínez-Murcia, A. J.
1999.
Phylogenetic positions of Aeromonas encheleia, Aeromonas popoffii, Aeromonas DNA hybridization group 11 and Aeromonas group 501.
Int. J. Syst. Bacteriol.
49:1403-1408[Abstract/Free Full Text].
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| | | | |
Maria José Figueras*
Josep Guarro
Departamento de Ciencias Médicas Básicas
Facultad de Medicina y Ciencias de la Salut
Universidad Rovira y Virgili
Sant Lorenzo 21
43201 Reus, Spain
|
| | | | |
Antonio Martínez-Murcia
Departamento de
Fisiología, Genética y
Microbiología
Universidad de Alicante
Aptdo. 99
03080 Alicante, Spain
Phone: 34-965903853
Fax: 34-965903943
E-mail: a.m.murcia{at}ua.es.
|
| |
AUTHOR'S REPLY |
I appreciate the interest in my recent study (6). After
carefully reading the comments, I do not believe that they affect or
alter the conclusions that I made. I also need to clarify that I never
stated that RFLP-PCR of the 16S rRNA gene should not be used for the
identification of Aeromonas species; I stated "However, verification of species identification with biochemical tests is still
appropriate for clinical diagnosis in light of the differences reported
in our study" (6). Indeed, we have used this approach in
combination with biochemical tests to identify environmental Aeromonas strains (11). Nor did I state or imply
that the 16S rRNA gene was responsible for changing biochemical
characteristics of the strains. The original contribution of Borrell et
al. (2) was clearly cited as a powerful method, and the
methodological differences are very clear and apparent to the reader,
even when only the figures are examined. The conclusion I would hope
that readers will take home from my paper is that analysis of both the
16S rRNA gene sequences and the biochemistry will produce a more
rigorous identification than either alone.
The most important discovery in the study was that three
Aeromonas veronii biovar sobria reference strains produced
an unexpected RFLP pattern that was not predicted from computer
analysis while four reference strains produced the expected pattern
(6). Thus, one can reasonably assume that not all A. veronii biovar sobria strains will produce the expected pattern.
Because I used the restriction endonuclease AluI, as did
Borrell et al. (2) in their study describing the use of
RFLP-PCR of the 16S rRNA gene for Aeromonas identification,
my results are applicable, relevant, and important to their study as
well. As long as AluI is included, even amplifying a larger
fragment of the 16S rRNA gene and digesting the DNA with a mixture of
the restriction endonucleases AluI and MboI will
not change the fact that some A. veronii biovar sobria reference strains identified by DNA-DNA hybridization will produce a
different pattern than expected.
The results of my study (6) underline the importance of
using a large enough set of well-characterized reference strains while
establishing an identification method, instead of using a few reference
strains and a collection of strains identified biochemically. In the
field of Aeromonas, we are aided by valuable previous
studies that have identified many strains by DNA-DNA hybridization and
characterized them by using biochemical tests (3, 9),
multilocus enzyme electrophoresis (1), and molecular methods
(7). I examined those studies and searched for reference strains that covered the range of diversity, especially within the
species of A. veronii biovar sobria.
There are four reasons why one might want to exercise caution when
using the 16S rRNA gene sequence for identifying Aeromonas species. First, Martinez-Murcia et al. (10) reported in 1992 that the actual sequence difference between Aeromonas
species can be very slight. For example, the Aeromonas trota
strain ATCC 49659T differs from the Aeromonas
caviae strain NCIMB 13016T by a single nucleotide and
the Aeromonas hydrophila strain ATCC 7966T
differed from the Aeromonas media strain ATCC
33907T by 3 nucleotides (10). Second, the
phylogenetic trees that have been constructed using the 16S rRNA gene
sequence suggest different degrees of relatedness than those inferred
from DNA-DNA hybridization studies (10). Third,
interestingly, it has been proposed that crossing-over of ribosomal
sequences has occurred several times in Aeromonas
(12). Fourth, it has been reported that for some bacteria,
intraspecific variation in the 16S rRNA is not uncommon (4)
and this was recently shown to occur in Aeromonas popoffii
(5). Anyone interested in rigorously identifying Aeromonas strains must be aware of these valid, published
concerns and their consequences when applying an approach that relies
on 16S rRNA gene sequences.
In response to some of the more detailed issues, such as which strains
to include under the species Aeromonas encheleia, we followed the recommendations from a study performed by investigators from three institutions that involved several approaches
(8); thus, there was no need to include additional
restriction enzymes to confuse the identification. In regards to the
critique of faints bands supposedly resulting from the incomplete
digestion of the samples, it should be noted that there is an
alternative explanation and that is that the strain analyzed carries
different 16S rRNA alleles that result in different RFLP patterns.
Finally, when I interpreted the results in regard to the species
identification, I specifically stated that I excluded single variants
(6). This was to ensure that if a strain is reclassified or
falsely classified it does not affect the final conclusions. In
addition, the identity of 10 reference strains with the unusual
patterns was verified by biochemical analysis to ensure that I had
received and analyzed the correct strains (6). All of this
was done in the interest of an accurate identification scheme.
As suggested in my report, my group has sequenced the 16S rRNA gene of
several strains with unexpected patterns and these results are
consistent and support the conclusions of our study (J. Graf and R. Troller, unpublished data). Hopefully, this will help to address some
of these issues in a scientific manner. RFLP-PCR of the 16S rRNA gene
is a valuable tool in the identification of Aeromonas
strains; however, it remains my concern that by relying solely on an
approach based on the 16S rRNA sequence, investigators may leave their
studies open to criticisms that could be avoided if they would apply
biochemical tests to independently verify their results.
| |
FOOTNOTES |
| |
REFERENCES |
| 1.
|
Altwegg, M.,
M. W. Reeves,
R. Altwegg-Bissig, and D. J. Brenner.
1991.
Multilocus enzyme analysis of the genus Aeromonas and its use for species identification.
Zentbl. Bakteriol.
275:28-45.
|
| 2.
|
Borrell, N.,
S. G. Acinas,
M. J. Figueras, and A. J. Martinez-Murcia.
1997.
Identification of Aeromonas clinical isolates by restriction fragment length polymorphism of PCR-amplified 16S rRNA genes.
J. Clin. Microbiol.
35:1671-1674.
|
| 3.
|
Carnahan, A. M., and S. W. Joseph.
1993.
Systematic assessment of geographically and clinically diverse aeromonads.
System. Appl. Microbiol.
16:72-84.
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| 4.
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Clayton, R. A.,
G. Sutton,
P. S. Hinkle, Jr.,
C. Bult, and C. Fields.
1995.
Intraspecific variation in small-subunit rRNA sequences in GenBank: why single sequences may not adequately represent prokaryotic taxa.
Int. J. Syst. Bacteriol.
45:595-599[Abstract/Free Full Text].
|
| 5.
|
Demarta, A.,
M. Tonolla,
A.-P. Caminada,
N. Ruggeri, and R. Peduzzi.
1999.
Signature region within the 16S rDNA sequences of Aeromonas popoffii.
FEMS Microbiol. Lett.
172:239-246.
|
| 6.
|
Graf, J.
1999.
Diverse restriction fragment length polymorphism patterns of the PCR-amplified 16S rRNA genes in Aeromonas veronii strains and possible misidentification of Aeromonas species.
J. Clin. Microbiol.
37:3194-3197.
|
| 7.
|
Huys, G.,
R. Coopman,
P. Janssen, and K. Kersters.
1996.
High-resolution genotypic analysis of the genus Aeromonas by AFLP fingerprinting.
Int. J. Syst. Bacteriol.
46:572-580[Abstract/Free Full Text].
|
| 8.
|
Huys, G.,
P. Kämpfer,
M. Altwegg,
R. Coopman,
P. Janssen,
M. Gillis, and K. Kersters.
1997.
Inclusion of Aeromonas DNA hybridization group 11 in Aeromonas encheleia and extended descriptions of the species of Aeromonas eucrenophila and A. encheleia.
Int. J. Syst. Bacteriol.
47:1157-1164.
|
| 9.
|
Kampfer, P., and M. Altwegg.
1992.
Numerical classification and identification of Aeromonas genospecies.
J. Appl. Bacteriol.
72:341-351[Medline].
|
| 10.
|
Martinez-Murcia, A. J.,
S. Benlloch, and M. D. Collins.
1992.
Phylogenetic interrelationships of members of the genera Aeromonas and Plesiomonas as determined by 16S ribosomal DNA sequencing: lack of congruence with results of DNA-DNA hybridizations.
Int. J. Syst. Bacteriol.
42:412-421.
|
| 11.
|
McLeod, E. S.,
Z. Dawood,
R. MacDonald,
M. C. Oosthuizen,
J. Graf,
P. L. Steyn, and V. S. Brözel.
1998.
Isolation and identification of sulphite- and iron reducing, hydrogenase positive faculative anaerobs from cooling water systems.
Syst. Appl. Microbiol.
21:297-305.
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| 12.
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Sneath, P. H. A.
1993.
Evidence from Aeromonas for genetic crossing-over in ribosomal sequences.
Int. J. Syst. Bacteriol.
43:626-629[Free Full Text].
|
| | | | |
Joerg Graf
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
|
Journal of Clinical Microbiology, May 2000, p. 2023-2025, Vol. 38, No. 5
0095-1137/00/$04.00+0
