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Antimicrobial Agents and Chemotherapy, October 1999, p. 2468-2472, Vol. 43, No. 10
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Decreased Azithromycin Susceptibility of
Neisseria gonorrhoeae Due to mtrR
Mutations
Leticia
Zarantonelli,1
Graciela
Borthagaray,1
Eun-Hee
Lee,2,3 and
William M.
Shafer2,3,*
National Center for Gonococcal Antimicrobial
Susceptibility Surveillance, Department of Microbiology, School of
Chemistry, Montevideo, 11800, Uruguay1;
Department of Microbiology and Immunology, Emory University
School of Medicine, Atlanta, Georgia 303322; and
Laboratories of Microbial Pathogenesis, Medical Research
Service, Veterans Affairs Medical Center (Atlanta), Decatur,
Georgia 303333
Received 7 June 1999/Returned for modification 15 July
1999/Accepted 29 July 1999
 |
ABSTRACT |
Single-dose azithromycin therapy has recently been used in Uruguay
for the treatment of uncomplicated gonococcal infections. As part of an
active surveillance study to monitor the emergence of antibiotic
resistance in gonococcal isolates, we examined the levels of
azithromycin susceptibility in 51 consecutive isolates obtained from
males with uncomplicated gonococcal urethritis. Isolates with decreased
susceptibility to azithromycin (MICs, 0.25 to 0.5 µg/ml) were common,
and these isolates often displayed cross-resistance to hydrophobic
antimicrobial agents (erythromycin and Triton X-100). Resistance to
erythromycin and Triton X-100 is frequently due to overexpression of
the mtrCDE-encoded efflux pump mediated by mutations in the
mtrR gene, which encodes a transcriptional repressor that
modulates expression of the mtrCDE operon. Accordingly, we
questioned whether clinical isolates that express decreased azithromycin susceptibility harbor mtrR mutations. Promoter
mutations that would decrease the level of expression of
mtrR as well as a missense mutation at codon 45 in the
mtrR-coding region that would result in a radical amino
acid replacement within the DNA-binding motif of MtrR were found in
these strains. When these mutations were transferred into
azithromycin-susceptible strain FA19 by transformation, the
susceptibility of gonococci to azithromycin was decreased by nearly
10-fold. The mtrCDE-encoded efflux pump system was
responsible for this property since insertional inactivation of the
mtrC gene resulted in enhanced susceptibility of gonococci to azithromycin. We conclude that the mtrCDE-encoded efflux
pump can recognize azithromycin and that the emergence of gonococcal strains with decreased susceptibility to azithromycin can, in part, be
explained by mtrR mutations.
| |
INTRODUCTION |
The emergence of strains of
Neisseria gonorrhoeae that express clinically significant
levels of resistance to penicillin and tetracycline is a global problem
that is particularly acute in South America (9, 25). The
loss of these relatively inexpensive antibiotics for the treatment of
gonococcal infections has resulted in the use of alternative treatment
regimens for effective clinical management of gonorrhea. For instance,
azithromycin has been shown to achieve high and prolonged levels in
tissues and cells, thereby allowing coverage against both gonococci and
Chlamydia trachomatis (7). For these reasons,
azithromycin has recently been used in Uruguay for the treatment of
uncomplicated gonococcal infections in males.
Azithromycin is a 15-membered azalide derived from erythromycin by the
replacement of the 9a carbonyl in the aglycone ring with a
methyl-substituted nitrogen (35). Despite its improved pharmacokinetic properties and bactericidal capacity against gonococci in vitro, there is now evidence that clinical isolates with decreased azithromycin susceptibility can be recovered. Thus, we recently described (5) that for 161 consecutive genital isolates from males with acute gonococcal urethritis, the MIC at which 90% of isolates are inhibited (MIC90) was 0.5 µg/ml. This result
suggested that azithromycin resistance in gonococci was present in
Uruguay and could pose significant problems for the effective
management of patients with gonococcal infections.
We have sought to determine the molecular basis for the decreased
azithromycin susceptibility in strains of gonococci isolated from our
patient population. In theory, resistance to azithromycin could arise
due to mechanisms similar to those for resistance to other macrolides
(10). These mechanisms include target site modification by
methylases encoded by erm genes (18, 20, 21) or
antibiotic inactivation due to the actions of different enzymes (1-4, 6, 17, 19, 22, 32) or the actions of efflux pumps
(16, 36, 43). Little is known about the mechanisms of
macrolide resistance in gonococci, but the recently described mtrCDE-encoded efflux pump has been suggested to be one
mechanism by which certain strains could express decreased
susceptibility to erythromycin (8, 12, 13, 23, 24, 33). This
efflux pump has been shown to mediate energy-dependent export of
structurally diverse hydrophobic antimicrobial agents. The
mtrCDE genes constitute a single transcriptional unit that
is negatively regulated by the product of the adjacent but divergent
mtrR gene. The mtrR gene product (MtrR) is a
transcriptional repressor that binds to a 31-nucleotide region that
encompasses the promoter element that drives transcription of
mtrCDE (23). Mutations within the mtrR-coding region or a 13-bp inverted repeat sequence
within the mtrR promoter can result in decreased
susceptibility of gonococci to erythromycin as well as other
hydrophobic agents. We questioned whether such mutations might explain
the decreased azithromycin susceptibility property that seems to be
increasing in prevalence in isolates obtained from infected individuals
in Uruguay. We now report that mtrR promoter and coding
region mutations occur frequently in strains that express decreased
susceptibility to azithromycin and that their presence can explain this property.
| |
MATERIALS AND METHODS |
Strains of N. gonorrhoeae used and growth
conditions.
A sample of 51 consecutive clinical isolates of
N. gonorrhoeae with no known epidemiologic relationship were
recovered from male patients with urethritis and were received in one
of our laboratories (the laboratory of G.B. and L.Z.) for national
(Uruguay) antimicrobial susceptibility surveillance studies. These
strains were collected in Uruguay during 1996 and 1997. All isolates
were identified as N. gonorrhoeae by conventional methods
and were frozen at 
70°C. Control strains (ATCC 49226, WHO III, WHO
V, WHO VII, FA19, KH12, and KH15) with known profiles of susceptibility to the antimicrobial agents used in this study were included. Strain
FA19 is considered a wild type with respect to the
mtrCDE-encoded efflux pump system (13). Strains
KH12 and KH15 are isogenic transformants of strain FA19. KH12 contains
an insertional mutation in the mtrC gene (12)
that renders it hypersusceptible to hydrophobic agents. Strain KH15 is
hyperresistant to hydrophobic agents and contains a single-base-pair
deletion in a 13-bp inverted repeat sequence within the promoter region
used for transcription of the mtrR gene (13).
Antimicrobial testing.
The MICs of azithromycin, crystal
violet, erythromycin, Triton X-100, and tetracycline were determined by
the agar dilution method as specified in the National Committee for
Clinical Laboratory Standards protocol (29). Penicillinase
production was tested with the chromogenic cephalosporin nitrocefin
(Oxoid Ltd., Basingstoke, United Kingdom). High-level resistance to
tetracycline was shown to be due to possession of tetM by
PCR as described previously (26). By using the criteria of
Morse et al. (28), strains that express high-level
resistance to hydrophobic agents due to the mtrCDE-encoded
system (Mtr phenotype) were identified with respect to their
susceptibilities to crystal violet (MIC, >1.0 µg/ml), erythromycin
(MIC, >2.0 µg/ml), and Triton X-100 (MIC, >2,000 µg/ml), as
determined by the method of Shafer et al. (37).
PCR amplification and DNA sequencing studies.
Chromosomal
DNA was prepared from test strains as described previously
(27). These DNA samples were used in PCRs to amplify the
mtrR gene, including the promoter region, as described
previously (23). The PCRs used the oligonucleotide primers
used by Lucas et al. (23): RPMAL#2
(5'-ACTGAAGCTTATTTCCGGCGCAGGCAGGG-3') and KH9#3
(5'-GACGACAGTGCCAATGCAACG-3'). The PCR products were
purified by use of the QIAquick PCR purification kit protocol supplied by the manufacturer. Automatic DNA sequencing was performed at the
Emory University DNA Sequencing Core Facility and used oligonucleotide primer RPMAL #2, KH9#1 (5'-GTCGCAGATACGTTGGAACAACG-3'), or
KH9#3.
Transformation studies.
In order to inactivate the
mtrCDE-encoded efflux pump system in strains that express
decreased susceptibility to azithromycin, four clinical isolates that
express the Mtr phenotype were transformed with 0.5 µg of chromosomal
DNA from strain KH12 (mtrC::Kmr) per
ml. The transformation reaction was as described previously (12), and recombinants were selected for their resistance to kanamycin (50 µg/ml). Insertional inactivation of mtrC in
transformants was confirmed by PCR with oligonucleotide primers KH9#2
(5'-CGTTTCGGGTCGGTTTGACG-3') and KH9#3. The mtrR
mutations in clinical isolate 9638 was introduced into strain FA19 by
transformation with chromosomal DNA and selection for
erythromycin-resistant transformants with 0.5 µg of erythromycin per
ml. The susceptibilities of the transformants to antimicrobial agents
along with that of the respective parental strain were determined as
described above.
Statistical analyses.
Linear correlations between
logarithmic values of the MICs of azithromycin, erythromycin, and
tetracycline were evaluated with the Pearson coefficient (r)
with EPI INFO Statistical Software.
| |
RESULTS AND DISCUSSION |
Antibiotic susceptibilities of gonococcal isolates.
We
determined (Table 1) the susceptibilities
of 51 consecutive isolates obtained from our patient population in
Uruguay to azithromycin, erythromycin, and tetracycline; due to the
high proportion (>50%) of 
-lactamase-producing gonococci in
Uruguay, penicillin susceptibility testing was not conducted.
Chromosomally mediated resistance to tetracycline was observed in
62.7% (32 of 51) of the isolates, and seven additional isolates
displayed higher levels of resistance to tetracycline (MIC, >16
µg/ml) due to a plasmid-borne tetM determinant. A high
proportion of isolates displayed a multiple-drug-resistance pattern.
There was a significant linear correlation between logarithmic values
of the MICs of tetracycline or the MICs of azithromycin and logarithmic
values of the MICs of erythromycin (r = 0.77 [P < 0.01] and r = 0.84 [P < 0.01], respectively) (data not shown).
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TABLE 1.
Susceptibilities of 51 clinical isolates of N. gonorrhoeae to tetracycline, erythromycin, and azithromycin
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|
Expression of Mtr phenotype in clinical isolates.
Using the
criteria of Morse et al. (28), we established that the Mtr
phenotype (elevated resistance to crystal violet and Triton X-100) was
expressed by nearly 50% of strains that displayed decreased
susceptibility to azithromycin, erythromycin, and tetracycline (Table
2). It is relevant to note that those
gonococci that displayed the Mtr phenotype also showed elevated
resistance to azithromycin, erythromycin, and tetracycline compared to
the level of resistance of those strains that did not display the Mtr
phenotype.
Identification of mtrR mutations in clinical
isolates.
Previous studies (13, 33, 38) revealed that
clinical isolates or laboratory-derived mutants that display resistance to hydrophobic agents frequently contain loss-of-function mutations in
the mtrR-coding sequence or a single-base-pair deletion in a
13-bp inverted repeat within the mtrR promoter. In order to determine whether the strains used in this study might contain mutations in the mtrR region, we randomly selected four
strains (strains 9608, 9633, 9638, and 9642) that exhibited decreased susceptibility to azithromycin and erythromycin for molecular analysis.
DNA was prepared from these isolates for PCR amplification of a nearly
1-kb sequence that encompassed the mtrR-coding and promoter
sequences. DNA sequencing studies of these PCR products revealed (Fig.
1) that all four strains contained the
previously described single-base-pair deletion in the mtrR
promoter region. This mutation is known to abrogate transcription of
mtrR while it enhances transcription of the
mtrCDE efflux pump operon (14, 23). These effects
result in high-level resistance to Triton X-100 (12, 38). In
addition to this promoter mutation, each strain contained a missense
mutation at codon 45, which results in a radical amino acid replacement
(Gly-45 to Asp-45) within the helix-turn-helix motif of MtrR (Fig. 1);
this mutation, in the absence of the promoter mutation, has been
observed previously (13, 33, 38) in other gonococcal
isolates that display intermediate levels of resistance to hydrophobic
agents.
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FIG. 1.
mtrR mutations in clinical isolates of
N. gonorrhoeae. Shown is the nucleotide sequence information
of the mtrR promoter region from strains FA19 and KH15 and
five clinical isolates that display decreased levels of azithromycin
susceptibility. The 10 and 35 hexamers are shown inside the boxes,
the presence of the 13-bp inverted repeat is shown with a line over the
sequence, the single-base-pair deletion is shown with a dash, and the
dinucleotide (TT) insertion in strain 9604 is underlined. Also
presented is the Gly-45 (G)-to-Asp-45 (D) replacement in the
helix-turn-helix motif of the MtrR proteins (210 amino acids in length)
of strains 9608, 9633, 9638, and 9642.
|
|
Transformation studies.
In order to establish whether the
mtrCDE-encoded efflux pump was responsible for the decreased
azithromycin susceptibility expressed by strains 9608, 9633, 9638, and
9642, we inactivated their mtrC genes by transformation with
the mtrC::Kmr determinant from strain
KH12 (12). Insertional inactivation of mtrC in
representative strains was confirmed by PCR analysis, as described in
Materials and Methods (data not shown). In all cases, representative
transformants of each clinical isolate displayed hypersusceptibility to
azithromycin, erythromycin, and Triton X-100 (Table
3). This result confirmed that an intact
mtrCDE operon was required for the hydrophobic agent
resistance profiles expressed by these clinical isolates.
In order to verify that the mutations within
mtrR are
important in determining the decreased levels of azithromycin
susceptibility,
we transformed strain FA19 with chromosomal DNA from
strain 9638.
We selected erythromycin-resistant transformants and
scored them
for cross-resistance to azithromycin and Triton X-100.
Representative
transformants (see FA19 [
mtrR-9838], Table
3) also had decreased
susceptibility to azithromycin and Triton X-100.
DNA sequencing
analysis of the
mtrR region from transformant
strain FA19 (
mtrR-9638)
revealed that it had acquired both
the single-base-pair deletion
in the promoter and the missense mutation
at codon 45 that are
present in donor strain 9638 (data not shown). It
is important
that the single-base-pair deletion in the 13-bp inverted
repeat
sequence of the
mtrR promoter was sufficient to
decrease the level
of susceptibility of gonococci to azithromycin by
nearly 10-fold.
This conclusion was drawn from a comparison of
azithromycin MICs
for isogenic strains FA19 and KH15 (Table
3), which
differ at
their
mtrR regions by only the single base pair in
the
mtrR promoter
(
13).
Identification of a novel mtrR promoter mutation.
The mtrR mutations in the clinical isolates described above
were identical to those reported for other strains of gonococci that
express resistance to multiple hydrophobic compounds (14, 39). During our screening of erythromycin-resistant clinical isolates, we noticed rare strains (e.g., strain 9604, Table 3) that,
compared to strain FA19, displayed a nearly 10-fold decrease in
susceptibility to azithromycin but only a 2-fold decrease in susceptibility to Triton X-100. We analyzed the importance of the
mtrCDE-encoded efflux pump in determining levels of
azithromycin and erythromycin susceptibility in strain 9604 by
constructing an insertional mutation within its mtrC gene
using donor DNA from strain KH12. A representative transformant of
strain 9604 bearing mtrC::Kmr
displayed significantly enhanced susceptibility to azithromycin, erythromycin, and Triton X-100 (Table 3). DNA sequence analysis of the
mtrR region of strain 9604 revealed that it contained a heretofore undescribed mutation in its promoter. This mutation represented a dinucleotide insertion (TT) into the 13-bp inverted repeat sequence (Fig. 1). This mutation would increase the spacing between the 10 and 35 hexamers from an optimal 17 nucleotides to an
unfavorable 19 nucleotides (14).
The combined genetic and molecular results obtained in this
investigation implicate the
mtrCDE-encoded efflux pump as a
mechanism
by which gonococci can express decreased susceptibility to
azithromycin.
Through mutations that are known (
13) to
abrogate transcription
of the gene (
mtrR) that encodes a
transcriptional repressor of
mtrCDE or loss-of-function
mutations in the repressor-encoding
gene, gonococci can overproduce the
MtrC-MtrD-MtrE efflux pump
to increase their capacity to export
hydrophobic agents. These
hydrophobic agents are structurally diverse
and include dyes (e.g.,
crystal violet), detergents (Triton X-100), and
drugs (erythromycin
and azithromycin). That azithromycin can be
recognized by this
efflux pump system is not surprising given its
structural similarity
to erythromycin. Other bacterial efflux pumps
(
30,
31,
34),
including the MexA-MexB-OprM pump of
Pseudomonas aeruginosa, which
displays similarity at the
amino acid level to the MtrC-MtrD-MtrE
pump of gonococci (
8,
13,
14,
40), can also recognize
macrolide
antibiotics.
Over the past decade, several studies have evaluated the efficacy of a
single, oral dose of azithromycin for treatment of
bacterial sexually
transmitted diseases, including gonorrhea.
Although a single, oral dose
of 1.0 g of azithromycin proved effective
for treatment of
uncomplicated chlamydial infections, treatment
of uncomplicated
gonococcal infections produced variable results
(
15,
41,
45). In this respect, failure of azithromycin therapy
for
patients with uncomplicated gonococcal urethritis has been
reported
(
11,
41,
42,
44,
46). The azithromycin MICs
for some
gonococcal strains isolated from patients who have failed
azithromycin
therapy were, in fact, similar to the MICs obtained
for the isolates
used in this investigation (0.25 to 0.5 µg/ml).
Our results
demonstrate that
mtrR mutations that are known to
result
(
12,
14,
23) in overproduction of the MtrC-MtrD-MtrE
efflux
pump are sufficient to result in these unfavorable azithromycin
MICs.
The acquisition of additional mutations outside of the
mtr gene complex that modify the azithromycin structure or its target
could
further enhance azithromycin resistance to levels that would
abolish
the effectiveness of azithromycin as an alternative antibiotic
in the
treatment of uncomplicated gonorrhea. Continued surveillance
and
monitoring of antibiotic susceptibility patterns are needed
to detect
the emergence of such
strains.
| |
ACKNOWLEDGMENTS |
We thank L. Pucko for help in manuscript preparation, Q. F. Carolina Marquez for the tetM amplification data, J.-R.
Dillon (Director of WHO/PAHO GASP-Americas Centre) for providing
susceptibility reference strains, and the external quality control program.
This work was supported by funds from the Program of Development in
Basic Sciences (PEDECIBA) from Uruguay (to G.B.) and by Public Health
Service grant AI-21150 (to W.M.S.) from the National Institutes of
Health. W.M.S. was supported by a Research Career Scientist award from
the Veterans Affairs Research Service.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, Emory University School of Medicine, 3001 Rollins Research Center, Atlanta, GA 30322. Phone: (404) 728-7688. Fax:
(404) 329-2210. E-mail: wshafer{at}emory.edu.
| |
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Antimicrobial Agents and Chemotherapy, October 1999, p. 2468-2472, Vol. 43, No. 10
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
