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Journal of Clinical Microbiology, August 2001, p. 3023-3024, Vol. 39, No. 8
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.8.3023-3024.2001
LETTERS TO THE EDITOR
Description of Mycobacterium heckeshornense sp. nov.
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LETTER |
In the November 2000 issue of the Journal of Clinical
Microbiology, Roth et al. (1) presented a paper with
the description of a new Mycobacterium species,
Mycobacterium heckeshornense, based on multiple isolation of
this species from one patient. Analyses of the biochemical and growth
properties of their strain revealed an obviously close relationship to
M. xenopi, with only three negative biochemical test results
(arylsulfatase, nicotinamidase, and pyrazinamidase activities)
discriminating this strain from M. xenopi. However, we
analyzed two strains with the identical 16S rRNA gene sequence (GenBank
accession number AJ243481; sequence submitted 28 June 1999 [http://www.ncbi.nlm.nih.gov/]) and found that the results for these
three tests were clearly positive, as they were for M. xenopi. Thus, it seems that these properties may vary among
clinical isolates of M. heckeshornense and cannot
conclusively be used to differentiate it from M. xenopi. These results once more emphasize that the description of a new species
should be based on a minimum number of strains, e.g., 5 to 10 strains.
A strong reason for regarding M. heckeshornense as a
separate species is its unique 16S rRNA gene sequence. Using this
sequence, the authors performed a similarity analysis that confirmed
the close relationship to M. xenopi. Yet, in spite of this
detailed analysis, the authors did not mention the entry (dated 25 October 1998) in the GenBank database of a sequence with the
accession number AF101243 (http://www.ncbi.nlm.nih.gov/).
Within the above-mentioned file a 249-bp fragment of the 5'
region of the ribosomal 16S rRNA, having a sequence identical to that
of the now newly described species M. heckeshornense, was
deposited and named M. sydneyiensis. This fragment comprised
one part (region A, according to the paper of Roth et al.
[1]) of the signature sequences that are widely used for
mycobacterial species identification. It is well known to the authors
of this letter that determination of only region A is not sufficient
for species identification. However, the existence of an identical
sequence within this region should have caused strong suspicion that
the same species was under examination and should at least have been
discussed in a paper aimed at the description of a new taxon. As of 24 July 2000, the entry AF101243 has contained the complete 16S rRNA,
which is 100% identical to the sequence of M. heckeshornense.
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REFERENCE |
| 1.
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Roth, A.,
U. Reischl,
N. Schönfeld,
L. Naumann,
S. Emler,
M. Fischer,
H. Mauch,
R. Loddenkemper, and R. M. Kroppenstedt.
2000.
Mycobacterium heckeshornense sp. nov., a new pathogenic slowly growing Mycobacterium sp. causing cavitary lung disease in an immunocompetent patient.
J. Clin. Microbiol.
38:4102-4107[Abstract/Free Full Text].
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| | | | |
Elvira Richter
Stefan Niemann
Sabine Ruesch-Gerdes
Forschungszentrum Borstel
National Reference Center for Mycobacteria
Parkallee 18
23845 Borstel, Germany
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| | | | |
Dag Harmsen
University of Wuerzburg
Institute for Hygiene & Microbiology
97080 Wuerzburg, Germany
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AUTHORS' REPLY |
It is more than obvious from our report on three M. heckeshornense strains isolated from two patients that this
species represents a look-alike of M. xenopi and that this
might cause problems in the differential identification of these two
scotochromogens if only phenon-specific characteristics were
considered. The comments of Richter et al. on two additional strains of
M. heckeshornense found in their laboratory, which yielded
positive results in tests for arylsulfatase, pyrazinamidase, and
nicotinamidase activities, point in this direction. Unfortunately, the
authors do not exactly specify what testing methods they used. It is
well known that results may differ when pyrazinamidase activity is
determined either by the agar method or by the method introduced by
Bönicke (4). M. heckeshornense shows a
very slow growth rate at 37°C (and even at 45°C), and we are well
advised to state how tests were performed, e.g., at higher incubation
temperatures or with higher inoculum sizes. More importantly (amidase
test results may indeed vary for this species), we want to emphasize
that interpretation of the arylsulfatase test results demands judicious
consideration of test incubation time and quantification of positive
results. Typically, about 60 and 99% of M. xenopi isolates
present a strongly positive result in the 3-day and the 10-day
arylsulfatase tests, respectively (1, 4). Thus, careful
attention to this test with respect to quantification may be the sole
biochemical clue that a subject strain with a negative or weakly
positive test result possibly represents a M. heckeshornense
isolate. However, with the introduction of tools of molecular biology,
we can now approach differential identification of bacteria in the
routine practice by highly precise methods applicable to all species, including those exhibiting identical patterns for phenotypic markers. Genetic methods have been improved with respect to simplification and
reliability, which makes them appropriate for routine use in clinical
laboratories today. M. heckeshornense can easily be identified using molecular methods, such as RFLP-PCR or automated sequencing (2).
Richter and colleagues claim that the description of new species should
be based on observation of at least 5 strains. What may not be
generally understood is that the process of resolving questions of
taxonomy is a complex one and goes through different stages in which
description, classification, and identification are considered
(3). Certainly, this approach, as it serves the needs of
the medical microbiology community, must achieve a reasonable
compromise between timeliness and breadth of coverage. It is nearly
needless to mention that clinical meaningfulness, paralleled by the
quest for precision, should be the key to timeliness (3).
The purpose of our article, then, was to describe a new species in a
timely fashion in view of the given clinical priority. If there is a
need to pursue the problem of separating this species from M. xenopi because both fit an identical biochemical test pattern, we
believe that it would be more fruitful to collect a large number of
strains (definitely more than five) in due course and to submit these
to a more comprehensive review using comparative analyses of
well-described tests. Interestingly, we have recently retrospectively
scanned our strain collection for atypical M. xenopi strains
and have found four isolates with a negative and one with a very weak
10-day arylsulfatase activity test result (the results for testing
of pyrazinamidase agar activity were variable, and testing of
nicotinamidase activity was not done). All of these strains were
identified as M. heckeshornense by means of genetic methods.
It is worth brief mention that two of these strains were isolated from
one patient with progressive cavitary lung disease and histologically
confirmed epitheloid cell granulomatosis. He had been treated in 1996 and 1997 for suspected but never proven tuberculosis. The first isolate
was obtained between the two treatment series in the mid-1990s, while
the second one was subcultured from one of the very recent multiple
smear-positive specimens submitted to our laboratory in April this year.
The comments on the missing citation of a previous entry in a sequence
database are appreciated because they draw our attention to a technical
problem that may flaw database searches. Of course, not mentioning this
sequence was not a matter of citation ethics (as would be the case for
citation amnesia or inertia); rather, it is explained by our choice of
search parameters. As we found out from a query at the National Center
for Biotechnology Information, the alignments attained after performing
the BLAST search may have received a high Expect value due to the
discrepancy in length between query (M. heckeshornense, 1.5 kbp) and hit (accession no. AF101243 gi:4092535, 249 bp [released in
January 1999]). Therefore, the alignment did not meet the Expect value
threshold for reporting output and was thus overlooked. Ultimately, 16S ribosomal DNA sequences with a length of at least 500 bp are regarded as a standard today, but workers dealing with ribosequencing should be
aware of the need to either perform additional searches with smaller
fragments of a long input sequence (preferably with those containing
variable regions of interest) or adapt search parameters.
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FOOTNOTES |
*
E-mail:
mikromau{at}zedat.fu-berlin.de
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REFERENCES |
| 1.
|
Kent, P. T., and G. P. Kubica.
1985.
Public health mycobacteriology a guide for the level III laboratory. U.S. Department of Health and Human Services Publication (CDC) 86-8230.
Centers for Disease Control, Atlanta, Ga.
|
| 2.
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Roth, A.,
U. Reischl,
A. Streubel,
L. Naumann,
R. M. Kroppenstedt,
M. Habicht,
M. Fischer, and H. Mauch.
2000.
Novel diagnostic algorithm for identification of mycobacteria using genus-specific amplification of the 16S-23S rRNA gene spacer and restriction endonucleases.
J. Clin. Microbiol.
38:1094-1104[Abstract/Free Full Text].
|
| 3.
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Telenti, A.
1998.
More on `What's in a name ... 'pragmatism in mycobacterial taxonomy.
Int. J. Tuberc. Lung Dis.
2:182-183[Medline].
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| 4.
|
Wayne, L. G.
1984.
Mycobacterial speciation, p. 25-65.
In
G. P. Kubica, and L. G. Wayne (ed.), The mycobacteria: a sourcebook. Microbiology series, vol. 15. Marcel Dekker, Inc, New York, N.Y.
|
| | | | |
Andreas Roth*
Harald Mauch
Nicolas Schönfeld
Robert Loddenkemper
Zentralklinik Emil von Behring
Lungenklinik Heckeshorn
D 14109 Berlin, Germany
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| | | | |
Udo Reischl
Ludmila Naumann
Institut für Medizinische Mikrobiologie
und Hygiene,
Universität Regensburg
D 93053 Regensburg, Germany
|
| | | | |
Reiner M. Kroppenstedt
Deutsche Sammlung von Mikroorganismen
und Zellkulturen
GmbH
D 38124 Braunschweig, Germany
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Journal of Clinical Microbiology, August 2001, p. 3023-3024, Vol. 39, No. 8
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.8.3023-3024.2001
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