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Antimicrobial Agents and Chemotherapy, October 2001, p. 2958-2960, Vol. 45, No. 10
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.10.2958-2960.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Two New Point Mutations at A2062 Associated with
Resistance to 16-Membered Macrolide Antibiotics in Mutant Strains of
Mycoplasma hominis
Pio Maria
Furneri,1,*
Giancarlo
Rappazzo,2
Maria Pia
Musumarra,1
Patrizia
Di
Pietro,1
Lucrezia S.
Catania,1 and
Lucia
Silvana
Roccasalva1
Dipartimento di Scienze Microbiologiche e
Scienze Ginecologiche1 and Dipartimento
di Biologia Animale,2 Università degli
Studi, Catania, Italia
Received 12 March 2001/Returned for modification 17 April
2001/Accepted 20 July 2001
 |
ABSTRACT |
We describe two mutants of Mycoplasma hominis PG-21
which show resistance to 16-membered macrolides but susceptibility to lincosamides, obtained by in vitro exposure to increasing doses of
josamycin. The 23S rRNA gene showed that each had a mutation (A2062G
and A2062T) corresponding to nucleotide 2062 in Escherichia coli, which was associated with the acquired phenotype.
 |
TEXT |
The genetic bases for
macrolide-lincosamide-streptogramin B group resistance have been
extensively studied for many bacteria (2, 4-6, 16, 18, 27, 29,
30, 32-34) and mycoplasmas (8, 14). In
Mycoplasma pneumoniae (14, 22), as well as in
other microorganisms, resistance to erythromycin has been associated with point mutations (A-to-G transition) in the loop of domain V of the
23S rRNA (2, 4-6, 8, 16, 18, 27, 29, 30, 32). Specific
residues within domain V of 23S rRNA are involved in the action of
macrolide-lincosamide-streptogramin antibiotics and chloramphenicol
(2-6, 8, 11, 14-18, 20, 27-30, 32). It is well known
that Mycoplasma hominis and many other mycoplasmas are
resistant to erythromycin but susceptible to 16-membered macrolides (josamycin and miocamycin) (1, 7-9, 19, 23) and
lincosamides (23). Recently the natural resistance of
M. hominis to erythromycin has been associated with a
guanine-to-adenine transition in position 2057 (Escherichia
coli numbering), located in the central loop of the 23S rRNA
domain V (8), and such a transition is present in
Mycoplasma flocculare and Mycoplasma
hyopneumoniae, which are also resistant to erythromycin
(24).
These features encouraged us to investigate the ability of josamycin to
select for resistance in M. hominis strains, in order to see
whether the acquired resistance pattern includes either macrolides only
or both macrolides and lincosamides and to establish a possible
relationship between the resistant phenotype and the appearance of
specific point mutations in the region coding for the peptidyl
transferase loop in the 23S rRNA gene.
The type strain M. hominis PG-21 was chosen for our
study. The strain was grown in SP-4 broth (pH 7.0) (7, 8, 21, 25,
26, 31). The MIC was determined by a broth microdilution assay
as previously described (7-9, 23).
For multistep selection for resistance, SP-4 broth medium containing
doubling concentrations of josamycin was inoculated with strain PG-21,
incubated at 37°C, and examined for growth each day. To test for the
development of resistance, 0.1 ml of the culture was withdrawn from
each tube every day and spread on an SP-4 agar plate containing 1 µg
of josamycin/ml. All the colonies growing on josamycin-agar were
challenged with increasing concentrations of the drug in broth (from 16 to 128 µg/ml). All selected mutants were single colony purified on
SP-4 agar-josamycin and drug-free SP-4 agar. Resistance to josamycin
was assayed after the microrganisms were repeatedly transferred to
antibiotic-free media.
Two resistant clones of the M. hominis PG-21 strain were
obtained by the selection procedure outlined above and were found to be
stably resistant. The resistance/susceptibility patterns for the two
strains, called PG-21/JR and PG-21/JR2, are shown in Table
1 (23).
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TABLE 1.
The resistance/susceptibility patterns of M. hominis PG-21/JR and PG-21/JR2 compared to that of M. hominis PG-21
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|
DNA was extracted and purified by standard methods. Oligonucleotide
primers were designed upon alignment of 23S rRNAs from a number of
closely related species (8). PCR amplification was
performed by standard methods. The cycling programs were as follows:
one cycle at 98°C for 10 min; 30 cycles of 95°C for 30 s,
53°C for 30 s, and 72°C for 30 s; and a final
elongation step at 72°C for 5 min. The DNA sequences were determined
with dye terminators.
Nucleotide sequence accession numbers.
The partial 23S rRNA
sequences of the M. hominis strains PG-21/JR and PG-21/JR2
have been submitted to GenBank under accession numbers AF184237 and
AF317663.
Since specific positions within the central loop of domain V of 23S
rRNA have been associated with the development of erythromycin
resistance in mycoplasma and in many other microrganisms, we examined
the sequence of this domain by amplifying the corresponding ribosomal
DNA gene of the two josamycin-resistant PG-21/JR strains. The
sequences
are shown in Fig.
1 aligned with the
corresponding sequences
from a number of related microorganisms and
Escherichia coli J01695 in order to number the nucleotide
positions. The G-to-A transition
at position 2057, already described
for
M. hominis as a naturally
occurring transition, helped
further in establishing the nucleotide
correspondence in domain V. In
the two josamycin-resistant derivatives,
two new mutations, an
A2062G transition and an A2062T transversion,
were observed in the
region coding for the peptidyl transferase
loop. These represent new
mutations, so far not reported in any
microorganism. In the original
electropherogram, the first appeared
as two coincident G and A peaks
while the second appeared as a
unique T peak (data not shown). In order
to exclude a sequence
artifact for the two coincident peaks seen, an
allele-specific
PCR experiment was performed (
35). Two
different primers were
designed whose 3' position matched the
nucleotide at position
2062 either for the mutated operon (endG,
5'-CCGCATCTAGACGAAAAGG-3';
endT,
5'-CCGCATCTAGACGAAAAGT-3') or for the wild type (endA:
5'-CCGCATCTAGACGAAAAGA-3').
These primers were used in
separate experiments in conjunction
with the reverse primer R1
(5'-CCTCCGTTACCTTTTAGGA-3') or R2
(5'-GGTCCTCTCGTACTAGAAG-3').
KCl was replaced with
(NH
4)
2SO
4, and the annealing
temperature
was increased to 59°C to increase stringency, keeping
unchanged
the remaining PCR parameters.

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FIG. 1.
Alignment (partial) of 23S rRNA genes from selected
organisms corresponding to the loop of the peptidyl transferase (domain
V). The nucleotides are numbered on the basis of the E. coli
sequence. Raised letters denote the following: a, GenBank accession no.
AF184237, M. hominis strain PG-21/JR 23S rRNA gene (partial
sequence); b, AF317663, M. hominis 23S rRNA gene (partial
sequence, strain PG-21/JR2); c, AF101242, M. hominis strain
PG-21 23S rRNA gene (partial sequence); d, AF131860, M. hominis strain CT-Mh1 23S rRNA gene (partial sequence); e,
AF131073, M. hominis strain CT-PAF 23S rRNA gene (partial
sequence); f, X68422, M. pneumoniae gene for 23S rRNA; g,
J01695, E. coli rRNA operon (rrnB).
|
|
As expected, the use of primers with different 3' ends gave rise to the
expected amplification product in the mutant strains,
whereas only the
wild-type-specific primer worked in the original
PG-21 strain (data not
shown). The heterozygous state of the A2062G
transition in the mutant
strain was thus definitively assessed,
while in the case of
transversion, an apparent homozygous state
was evidenced by both the
electropherogram and allele-specific
PCR. Control experiments, done on
pools of either uninduced or
induced (resistant) strain cultures using
oligonucleotides designed
to match all possible mutations at position
2062, failed to detect
any other mutation at that position (data not
shown).
The involvement of domain V in the mechanisms of resistance has been
demonstrated by chemical footprinting studies (
2,
17),
which showed that the reactivity of certain purines within
domain V was
specifically altered by macrolides (erythromycin
and carbomycin, a
16-membered macrolide), lincosamides, vernamicin
B, chloramphenicol, or
azalides. Moazed and Noeller (
17) incubated
70S ribosomes
together with antibiotics and showed direct protection
of both A2058
and A2059 by both erythromycin and carbomycin against
derivatization by
dimethyl sulfate; carbomycin additionally protected
position A2062. Of
the three protectable adenine residues, vernamycin
B, a streptogramin,
protected A2062 but not A2058 or A2059. Moreover,
clindamycin protected
both A2058 and A2059, whereas lincomycin
protected only A2058
(
4-5).
The role of the adenine residue at position 2062 is not fully defined.
An A2062C transversion, associated with chloramphenicol
and with
linezolid resistance in
Halobacterium halobium, was found
at
the same position (
12,
15). More recently, an A2062C
transversion
has been associated with 16-membered macrolide and
streptogramin
resistance in
Streptococcus pneumoniae
(
3). While no drug effects
could be directly attributed to
the A2062 site, raising the possibility
that such an effect, if it
exists, may be attributed to ribosomal
protein (
20), A2062
was shielded by virginiamycin M (a streptogramin
A) in a mutant strain
resistant to virginiamycin S (a streptogramin
B) (
28). The
aforementioned features may explain why our mutant
strains became
resistant to 16-membered macrolides due to mutation
at position 2062 and retained their susceptibility to lincomycin
and clindamycin.
An analysis of our mutants suggests that A2062
is incompatible with
16-membered macrolide resistance; however,
since a role of A2062 in
resistance has been questioned by some
authors, as shown above, we
hypothesize that A2062 incompatibility
with macrolide resistance could
be associated with species-specific
sequence context, particularly with
the naturally occurring G2057A
transition. Further studies will be
necessary to confirm this
hypothesis.
Since there are two rRNA operons in
M. hominis (
10,
13), a new mutation in any operon should be present in a
heterozygous
state. Under selective pressure the mutant
"allele" should behave
as dominant, allowing the strain to
overcome antibiotic inhibition.
The effect of antibiotics on the rRNA
stability in both wild-type
and mutant strains should also be taken
into
account.
 |
ACKNOWLEDGMENTS |
This work was supported by grants from University of Catania (1997 to 2000).
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Dipartimento di
Scienze Microbiologiche e Scienze Ginecologiche, Università di
Catania, Via Androne 81, 95124 Catania, Italy. Phone: 39 095316038. Fax: 39 095312798. E-mail: furneri{at}mbox.unict.it.
 |
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Antimicrobial Agents and Chemotherapy, October 2001, p. 2958-2960, Vol. 45, No. 10
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.10.2958-2960.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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