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Journal of Clinical Microbiology, October 2000, p. 3910-3911, Vol. 38, No. 10
0095-1137/00/$04.00+0
LETTERS TO THE EDITOR
Mycoplasma fermentans, M. hominis, and M. hyorhinis Inhibit Infectivity and Growth of Chlamydia
trachomatis and C. pneumoniae in HEp-2 Cells
 |
LETTER |
We read with great interest the data reported by Castilla and
Wadowsky about the elimination of a Mycoplasma hominis-like mycoplasma from the TW-183 strain of Chlamydia pneumoniae
and its lack of effect on chlamydial infection of HEp-2 cells
(1).
We recently described detection of Mycoplasma fermentans, M. hominis, and M. hyorhinis in several strains of
C. trachomatis and C. pneumoniae, the successful
elimination of Mycoplasma from chlamydial culture by the
antibiotic mupirocin, and the profound effect of mycoplasmal
contamination on chlamydial infectivity and growth in HEp-2 cells
(2). We would like to bring to the attention of your
readership several important different or additional points.
First, in contrast to the detergent Igepal CA-630 suggested by Castilla
and Wadowsky for elimination of Mycoplasma, the antibiotic mupirocin enabled the effective elimination of Mycoplasma
without affecting the growth of C. trachomatis or
C. pneumoniae (2). Second, we would strongly
recommend the use of PCR instead of culture for analysis of
Mycoplasma contaminations, since we repeatedly observed
mycoplasmal contamination as determined by specific PCR in mycoplasmal
culture-negative chlamydial preparations (B.K.-O., unpublished
observation). In addition, sequencing of the PCR product allows species
identification, information which may be important to track down the
potential contaminating source (2, 4). Third, the point that
we find most intriguing in the paper by Castilla and Wadowsky is the
lack of effect of mycoplasmal contamination on chlamydial infectivity.
In contrast to their observation, we found a profound inhibitory effect
of mycoplasmal contamination on chlamydial infectivity and growth
(2). Similar results showing M. hyorhinis
contamination of C. psittaci were recently published (3). Of course the differing mycoplasmal species present or differences in the culture media in those chlamydial cultures may
explain this lack of effect. However, when we cocultivated M. hominis with chlamydiae, we also observed a strong inhibition of
chlamydial growth (B.K.-O., unpublished observation). Another explanation for the observation of Castilla and Wadowsky may be that
although the chlamydiae used in their studies were Mycoplasma culture negative, those chlamydiae might have still contained Mycoplasma that was detectable only by PCR. When we
determined the ratio of chlamydial organisms, i.e., the number of
elementary bodies per inclusion-forming unit for both mycoplasma-free
and mycoplasma-contaminated chlamydiae, this ratio was 10 times higher for the mycoplasma-contaminated chlamydiae; that is, although the
numbers of inclusion-forming units were identical, the infectivity determined per chlamydial organism was much lower for the
mycoplasma-contaminated chlamydiae (2). By using chlamydiae
standardized for their infectivity, i.e., the same number of
inclusion-forming units, an additional inhibitory effect of the
mycoplasma added to the already latently infected chlamydiae might have
been missed in their experiments, especially since Castilla and
Wadowsky analyzed chlamydial growth already after 3 days of culture. In
our experience, the inhibitory effect of Mycoplasma on
chlamydial growth became most evident after 7 days of cocultivation
(B.K.-O., unpublished observation).
| |
FOOTNOTES |
*
Phone:
49-511-532-2190 Fax: 49-511-532-5841 E-mail:
Kuipers.Jens{at}MH-Hannover.DE
| |
REFERENCES |
| 1.
|
Castilla, E. A., and R. M. Wadowsky.
2000.
Effect of a Mycoplasma hominis-like mycoplasma on the infection of HEp-2 cells by the TW-183 strain of Chlamydia pneumoniae.
J. Clin. Microbiol.
38:861-862[Abstract/Free Full Text].
|
| 2.
| Krauße-Opatz, B., P. Dollmann, H. Zeidler, J. G. Kuipers, and L. Köhler. Frequent contamination of
Chlamydia trachomatis and Chlamydia pneumoniae
strains with mycoplasma. Biological relevance and selective
eradication of mycoplasma from chlamydial cultures with mupirocin. Med.
Microbiol. Immun., in press.
|
| 3.
|
Van Nerom, A.,
R. Ducatelle,
G. Charlier, and F. Haesebrouck.
2000.
Interaction between turkey monocytes and avian Chlamydia psittaci in the presence of Mycoplasma sp.: the importance of nitric oxide.
Dev. Comp. Immunol.
24:417-432[Medline].
|
| 4.
|
Wirth, M.,
E. Berthold,
M. Grashoff,
H. Pfützner,
U. Schubert, and H. Hauser.
1994.
Detection of mycoplasma contaminations by polymerase chain reaction.
Cytotechnology
16:67-77[CrossRef][Medline].
|
| | | | |
Birgit Krauße-Opatz
Petra Dollmann
Henning Zeidler
Lars Köhler
Jens G. Kuipers*
Abt. Rheumatologie
Medizinische Hochschule Hannover
Carl-Neuberg-Str.
1
30625 Hannover, Germany
|
| |
AUTHORS' REPLY |
We read with interest the letter by Krauße-Opatz et al. on the
comparisons of their study and our study on Mycoplasma
contamination of C. pneumoniae stock strains. As indicated
in their letter, stock suspensions of C. pneumoniae are
often contaminated with various Mycoplasma species. Our
study demonstrated the presence of a Mycoplasma hominis-like
Mycoplasma in the TW-183 strain of C. pneumoniae,
obtained from the American Type Culture Collection (ATCC), by
microbiologic culture. Similarly, another group (1) reported
the isolation of Mycoplasma by culture from two additional and independent sources of the TW-183 strain. These results suggest that Mycoplasma contamination of the TW-183 strain is
widespread. In contrast, a report by Krauße-Opatz et al.
(2) did not identify any Mycoplasma spp. in the
TW-183 strain derived from another source on the basis of testing with
a nested PCR-based assay that amplifies targets within ribosomal DNA.
One possible explanation for the different findings is that their
TW-183 strain is indeed mycoplasma-free. Alternatively, there may be
strains of Mycoplasma that are not detected with the
PCR-based assay. Collectively, these studies would suggest that it may
be prudent to evaluate stock cultures of C. pneumoniae for
Mycoplasma contamination using both a sensitive culture
assay and a PCR-based assay. However, the application of PCR-based
assays that amplify ribosomal DNA targets for detection of
Mycoplasma contamination in C. pneumoniae stock
cultures should be interpreted with some caution since cross-reacting sequences have been found in the ribosomal DNA of Mycoplasma
and C. pneumoniae (3). Although it appears that
endonuclease treatment of the ribosomal DNA amplicons and analysis of
the digests are helpful in identifying Mycoplasma
contamination, in our opinion isolation of the contaminant by culture
should be considered as definitive evidence of contamination. Isolation
of the Mycoplasma contaminant by culture also permits
assessment of the effect of the "actual" contaminant on the
infectivity of the C. pneumoniae strain. It is likely that
the TW-183 strain of C. pneumoniae used in many laboratories
has been contaminated for some time with Mycoplasma. Serial
passages of the TW-183 strain would have resulted in the simultaneous
passage of the autochthonous Mycoplasma, a practice that may
have selected for chlamydiae and mycoplasma with traits of coexistence.
In the study by Krauße-Opatz et al. (2), exogenous strains
of Mycoplasma were coincubated with a strain of C. pneumoniae, which was a different strain from the one used in our
study. These differences could have contributed to the difference
observed between the two studies on the effect of the various
mycoplasmas on the infectivity of the chlamydiae. We do not agree that
7 days of incubation are necessary to observe an effect with the
M. hominis-like Mycoplasma as stated by
Krauße-Opatz. During the 72-h incubation period of our cocultures, a
dramatic increase in the number of CFU of the M. hominis-like Mycoplasma per milliliter occurred in the
cocultures (4). Despite their statement on the importance of
7 days of incubation, the study by Krauße-Opatz et al. (2)
enumerated inclusions of C. pneumoniae in HEp-2 cells at day
4. We choose a 72-h incubation period since this incubation period is
commonly used in many laboratories. We also did not observe any
difference in the size of the C. pneumoniae inclusions
resulting from coincubation of the chlamydiae and the M. hominis-like Mycoplasma in HEp-2 cells and those
resulting from incubation of the Mycoplasma-free chlamydiae
in HEp-2 cells, a finding that is consistent with the view that the
number of elementary bodies per inclusion was the same under both conditions.
The finding of Krauße-Opatz et al. (2) that treatment of
C. pneumoniae stock suspensions with mupirocin effectively
eradicates Mycoplasma contamination is a valuable
contribution especially in view of our experience using Igepal
CA-630. We found that Igepal CA-630 has considerable activity against
C. pneumoniae and provides a relatively small window of
opportunity for eradicating Mycoplasma contamination. On the
other hand, our treatment schedule for Igepal CA-630 is relatively
simple, requiring a single 10-min exposure, compared to the mupirocin
treatment schedule, which utilizes incubation for up to 2 weeks with
frequent changes with mupirocin-containing medium.
| |
ACKNOWLEDGMENTS |
We thank B. Krauße-Opatz et al. for their thoughts on our study
and also acknowledge the importance of their new and important contributions.
| |
FOOTNOTES |
| |
REFERENCES |
| 1.
|
Huniche, B. S.,
L. T. Jensen,
S. Birkelund, and G. Christiansen.
1998.
Mycoplasma contamination of Chlamydia pneumoniae isolates.
Scand. J. Infect. Dis.
30:181-187[CrossRef][Medline].
|
| 2.
| Kraue-Opatz, B., H. Z. P. Dollmann, J.G., J. Kuipers, and
L. Kohler. Frequent contamination of Chlamydia
trachomatis and Chlamydia pneumoniae strains with
mycoplasma. Biological relevance and selective eradication of
mycoplasma from chlamydial cultures with mupirocin. Med. Microbiol.
Immun., in press.
|
| 3.
|
Messmer, T. O.,
C. M. Black, and W. L. Thacker.
1994.
Mycoplasma contamination of chlamydiae isolated from clinical specimens.
APMIS
102:793-796[Medline].
|
| 4.
|
Castilla, E. A., and R. M. Wadowsky.
2000.
Effect of a Mycoplasma hominis-like mycoplasma on the infection of HEp-2 cells by the TW-183 strain of Chlamydia pneumoniae.
J. Clin. Microbiol.
38:861-862.
|
| | | | |
Elias A. Castilla
The Cleveland Clinic Foundation
Department of Anatomic
Pathology
Cleveland, Ohio 44195
|
| | | | |
Robert M. Wadowsky
Department of Pathology
Children's Hospital of
Pittsburgh
Pittsburgh, Pennsylvania 15213
|
Journal of Clinical Microbiology, October 2000, p. 3910-3911, Vol. 38, No. 10
0095-1137/00/$04.00+0
