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Journal of Clinical Microbiology, February 1999, p. 442-444, Vol. 37, No. 2
Department of Mycobacteriology, Statens Serum
Institut, 2300 Copenhagen S, Denmark
Received 29 July 1998/Returned for modification 22 September
1998/Accepted 23 October 1998
Recently a novel insertion element, IS1245, has been
described and suggested for use as a probe in restriction fragment
length polymorphism studies of Mycobacterium avium strains.
An important issue in this context is the stability of the insertion
element. We analyzed single colonies of M. avium cultures
and found frequent small one- to two-band changes. However, following
repeated in vitro passages over 1 year, similar one- to two-band
changes were observed in the IS1245 patterns of only six
M. avium strains investigated.
Mycobacteria belonging to the
Mycobacterium avium complex (MAC) have long been known to be
potentially pathogenic for humans, with pulmonary infections and
lymphadenitis in small children as the most common clinical
presentations (6). However, MAC is also capable of causing
disseminated infection in severely immunocompromised individuals, and
due to the human immunodeficiency virus (HIV) pandemic, an increase in
these infections has been observed (5). As many as 25 to
40% of the HIV-infected patients contract MAC infections when their
CD4 cell counts reach a level of 50 × 109 to 100 × 109/liter (8). This circumstance has created
an increased interest in the epidemiology of MAC, with special
attention being given to routes and sources of infection. Studies of
these topics are dependent on reliable methods for strain
differentiation. A considerable number of genetic tools have become
available during recent years, e.g., pulsed-field gel electrophoresis
(2, 7), PCR-based methods (10), and restriction
fragment length polymorphism (RFLP) using different insertion elements
(3, 12). Pulsed-field gel electrophoresis has been
demonstrated to be a useful technique (13, 17, 18). However,
RFLP has already been established in many mycobacteriology laboratories
as the method of choice for the genotyping of Mycobacterium
tuberculosis. A proposal for the standardization of RFLP of
M. avium using the recently described insertion sequence
IS1245 has been elaborated (16). Experiences in
the studies of molecular epidemiology of M. tuberculosis
show that such a standardization is of great value since it allows comparison of data obtained in different laboratories (4,
15).
Knowledge of the stability of the insertion element in vivo and in
vitro is crucial for interpretation of the results. With the original
purpose to investigate the possible occurrence of polyclonal M. avium infections in HIV-infected patients, we undertook a study of
three AIDS patients who all had disseminated M. avium infections. The study was based on the hypothesis that polyclonal colonization of the intestine might precede translocation of one or a
very limited number of infectious organisms leading to monoclonal infection. Species identification was performed by DNA-RNA
hybridization using Accu-Probe (Gen-Probe Inc., San Diego, Calif.). A
total of eight cultures from the three patients were collected. All patients had positive cultures from stool and blood. One patient had
one additional isolate cultured from a liver biopsy specimen, and
another patient had M. avium cultured from sinus
frontalis. From each specimen, three colonies were subcultured, and
from the resulting 24 cultures purified single colonies were obtained. Four single colonies from each culture were further subcultured for
RFLP analysis, for a total of 96 bacterial clones (Fig.
1). DNA for RFLP analyses was obtained
from 87 isolates. RFLP analyses of these isolates were performed as
previously described (14) with the only minor alterations
that gels were run longer in order to obtain better separation of the
hybridization bands. In brief, DNA was extracted and digested with
PvuII. After electrophoresis on agarose gels, the digested
DNA was transferred to nylon membranes (Hybond N+; Amersham) and probed
with a chemiluminiscence-labelled 427-bp sequence of IS1245
generated by PCR as previously described (3). Colonies from
various locations on the same patient in all cases exhibited almost
identical IS1245 patterns. Yet differences affecting one to
two bands were observed in 5 of 33 isolates from patient A, in 4 of 34 isolates from patient B, and in 11 of 20 isolates from patient C. An
example of results from patient C is shown in Fig.
2. As is apparent from the autoradiogram,
some hybridization bands appeared weaker than others. Similar weak bands have been observed in isolates analyzed in a previous study (1) and might be due to cross-hybridization with
IS1311 as previously described (12).
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Stability of Insertion Sequence IS1245,
a Marker for Differentiation of Mycobacterium avium
Strains
ABSTRACT
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FIG. 1.
Schematic presentation of the preparation of
single-colony cultures from three patients.
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FIG. 2.
Autoradiogram showing PvuII-digested
IS1245 patterns of purified single colonies from patient C. Lanes: 1 to 7, RFLP patterns of single colonies from stool; 8 to 16, RFLP patterns of single colonies from blood. The arrows indicate
positions where changes occur.
To further explore whether these small differences among the clones
indicated a high degree of instability of IS1245, we decided to investigate the in vitro stability of the insertion element. A total
of six M. avium cultures were collected, four of which had
been analyzed by RFLP in another study (1) and two of which were single-colony isolates (Table 1).
These six isolates were initially subcultured in Dubos Tween 80 medium.
From November 1996 until November 1997, they were subcultured 33 times
in liquid media at intervals of 1 to 2 weeks. The first and last
subcultures from each isolate were analyzed by RFLP. As shown in Fig.
3, the isolates exhibited identical or
almost identical IS1245 patterns following the subcultures.
In four cases, differences in one or two bands could be observed.
Considering the fact that human M. avium isolates cultured
in Denmark almost exclusively exhibit IS1245 patterns with a
high number of bands (1), one- to two-band differences were
considered minor changes. Because the driving force of the observed
instability of the IS1245 RFLP pattern is not fully
understood, we do not know whether M. avium strains containing only a few IS1245 elements will exhibit the same
degree of instability.
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Previous studies have demonstrated that the IS1245 pattern exhibited by isolates cultured from the same patient over time is quite stable (9, 11). The degree of changes in the IS1245 patterns of such serial isolates seems to be comparable to those observed in serial M. tuberculosis isolates cultured from the same patient (19). This indicates that the stability of the insertion element in vivo is good. In this study, an attempt to uncover possible polyclonal infections failed but demonstrated several small one- to two-band differences among single colonies from the same isolate. Similar observations were made by Pestel-Caron and Arbeit (9), who recently described the same biological variations within the same culture. It was further demonstrated that no changes in the IS1245 pattern could be observed following subcultures on solid media over several months. In this study, only one- to two-band variations were observed in four of six M. avium strains subcultured in liquid media 33 times over a period of 1 year. Since small changes, however, do seem to occur, the finding of indistinguishable IS1245 banding patterns among M. avium strains must consequently be a strong indicator that these strains are actually related. This study therefore concludes that the stability of IS1245 is sufficient to recommend its use as a tool for differentiation of M. avium strains.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Mycobacteriology, Statens Serum Institut, Artillerivej 5, 2300 Copenhagen S, Denmark. Phone: 45 3268 3705. Fax: 45 3268 3871. E-mail: jba{at}ssi.dk.
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REFERENCES |
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