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Antimicrobial Agents and Chemotherapy, August 1998, p. 2103-2105, Vol. 42, No. 8
Division of AIDS, STD, and TB Laboratory
Research, National Center for Infectious Diseases, Centers for Disease
Control and Prevention, Atlanta, Georgia,1 and
Neisseria Reference Laboratory, Department of Medicine,
University of Washington, Seattle, Washington2
Received 20 January 1998/Returned for modification 8 April
1998/Accepted 20 May 1998
Of 65 ciprofloxacin-resistant, clinical isolates of Neisseria
gonorrhoeae, 5 isolates exhibited ParC mutations previously undescribed in the gonococcus. For isolates containing two ParC mutations (the Ser-87 Fluoroquinolones are frequently used
in the treatment of gonorrhea, with ciprofloxacin and ofloxacin used as
primary treatment regimens in a number of countries. Recently, a number
of gonococcal strains with decreased susceptibility or clinically
significant resistance to the Centers for Disease Control and
Prevention (CDC)-recommended doses of fluoroquinolones have been
isolated (2, 11). A number of studies have been
performed to determine the mechanism of fluoroquinolone resistance in
Neisseria gonorrhoeae (1, 3, 4, 6, 18, 19). The
most prevalent mechanisms contributing to fluoroquinolone resistance in
the gonococcus involve mutations in the quinolone resistance-determining region of gyrA and the analogous
region of the parC locus on the chromosome. These mutations
result in altered GyrA and ParC proteins (1, 4, 6, 18).
These altered proteins can no longer be bound by the fluoroquinolone, and therefore, the drug is unable to inhibit DNA replication and the
bacterium becomes less susceptible or resistant. The level of
susceptibility appears to correlate to the location and number of
mutations present (6). This mechanism is analogous to those observed in Escherichia coli and other bacteria (9,
20). However, unlike E. coli, mutations in the
N. gonorrhoeae gyrB gene do not appear to have a significant
impact on fluoroquinolone resistance (7), and no
parE homolog has been described.
Deguchi et al. (4-8) have studied the effects that these
mutations have on the level of fluoroquinolone resistance seen in various clinical gonococcal isolates. The mutations reported to date
have occurred in amino acids Ser-91 and Asp-95 of GyrA and amino acid
Asp-86, Ser-87, Ser-88, or Glu-91 of ParC. The order in which
mutations occur and result in increased fluoroquinolone MICs for
gonococcal strains follow a fairly predictable order: mutations in Ser-91 and/or Asp-95 of GyrA result in decreased susceptibility to ciprofloxacin (ciprofloxacin MICs, 0.125 to 0.5 µg/ml). On the other hand, clinically significant resistance to
CDC-recommended fluoroquinolone regimens (MIC, Eighty-three clinical isolates of N. gonorrhoeae were
obtained from commercial sex workers during the course of a study on fluoroquinolone resistance in the Republic of the Philippines in 1996. All isolates exhibited decreased susceptibility to ciprofloxacin (MICs,
Of the 83 isolates characterized, five isolates (Table
1) exhibited mutation patterns not
described previously (6). PCR and restriction endonuclease
analysis showed that all five isolates contained GyrA mutations at both
Ser-91 and Asp-95. Three isolates (isolates RP96-70, RP96-c1-003, and
RP96-c2-001) contained two ParC mutations at Ser-87 and Glu-91. Two
other isolates (isolates RP96-46 and RP96-61) contained no mutations in
ParC detectable by PCR and restriction endonuclease analysis. All five
isolates were further characterized by sequencing of the region of the ParC gene known to be involved in fluoroquinolone resistance. Primers
for sequencing were designed to produce a 300-bp fragment, representing
amino acids 60 through 139, by PCR. The primers used were a
forward-synthesis 21-base oligonucleotide with the sequence 5'-GTT TCA
GAC GGC CAA AAG CCC-3' and a reverse-synthesis 21-base oligonucleotide
with the sequence 5'-GGA CAA CAG CAA TTC CGC AAT-3'. PCR products were
purified for sequencing with the High Pure PCR Product Purification Kit
(Boehringer Mannheim). Sequencing reactions were performed in 20-µl
volumes with ABI Prism dRhodamine Terminator Cycle Sequencing Ready
Reaction Kit reagents (Applied Biosystems Division of the Perkin-Elmer
Corporation, Foster City, Calif.) containing 8.0 µl of Terminator
Ready Reaction Mix, 2 µl of PCR product template (approximately 75 ng), 3.2 µl of 1 µM primer, and 6.8 µl of sterile, deionized
water. Reactions were cycle sequenced on a GeneAmp PCR System 2400 (Perkin-Elmer) in 25 cycles of denaturation at 96°C for 10 s,
annealing at 50°C for 5 s, and extension at 60°C for 4 min.
Spin column purification with Centrisep Columns (Princeton Separations,
Adelphia, N.J.) was used to remove excess dye terminators and to purify
the reaction mixtures before electrophoresis. Electrophoresis of the
extension reaction mixtures was performed on the ABI Prism 377 DNA
Sequencer (Applied Biosystems Division of Perkin-Elmer Corporation)
with a 4% polyacrylamide gel (19:1 acrylamide:bis acrylamide; Bio-Rad
Laboratories, Hercules, Calif.).
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification of Novel Mutation Patterns in the
parC Gene of Ciprofloxacin-Resistant Isolates of
Neisseria gonorrhoeae
ABSTRACT
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Ile and Glu-91Gly mutations and the
Gly-85Cys and Arg116Leu mutations) the MICs of ciprofloxacin (8.0 to 64.0 µg/ml) were higher than those for the isolate containing the
single ParC mutation (Arg-116Leu; MIC, 1.0 µg/ml).
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1.0 µg of
ciprofloxacin per ml) appears to require one or more mutations in the
ParC gene, in addition to both gyrA mutations. However, this
order of mutations is not absolute and a number of gyrA and
parC mutation patterns have been described. In this paper,
we report the characterization of gonococcal isolates exhibiting
clinically significant resistance to ciprofloxacin (MICs, 1.0 to 64.0 µg/ml) in which the parC mutations differ either in number
or in location from those described previously.
0.125 µg/ml) and were characterized by auxotype, serovar, and
antibiotic sensitivities as described previously (10, 12-14, 17). Of these isolates, 65 were ciprofloxacin resistant (MIC range, 1.0 to 64.0 µg/ml). The presence of gyrA and
parC mutations was determined by PCR and restriction
endonuclease reaction analysis as described by Deguchi et al. (5,
8). Regions of gyrA and parC were amplified
by PCR after extraction of the chromosomal DNA of each gonococcal
strain. PCR amplification consisted of 35 cycles of denaturation at
94°C for 60 s, annealing at 52°C for 50 s, and extension
at 72°C for 50 s in reaction aliquots of 50 µl. Each PCR
mixture contained 5 µl of 10× PCR buffer (Boehringer Mannheim,
Indianapolis, Ind.), 1 µl of a deoxynucleoside triphosphate stock
solution (containing dATP, dCTP, dGTP, and dTTP each at a concentration
of 10 mM; Boehringer Mannheim), 0.5 µl of Taq DNA
polymerase (5 units/µl; Boehringer Mannheim), 2.5 µl of each of the
appropriate primers (1 mM), 28.5 µl of sterile water, and 10 µl of
template DNA. PCR primers specific for gyrA and
parC were the same as those described by Deguchi et al.
(8) and were synthesized at the National Center for
Infectious Diseases core facility at CDC in Atlanta, Ga. For each
isolate 10 µl of PCR product was restricted with endonuclease
HinfI for gyrA or EcoRI,
PstI, SalI, or HinfI for
parC (Boehringer Mannheim) and was visualized after
electrophoresis on a 6.5% polyacrylamide gel.
TABLE 1.
Characteristics of six clinical isolates of N. gonorrhoeae containing novel mutations in the
ParC genea
Sequence analysis confirmed the presence of two mutations in the ParC
genes of isolates RP96-70, RP96-c1-003, and RP96-c2-001 that
corresponded to amino acid changes at Ser-87Ile and Glu-91Gly. Sequence analysis of isolates RP96-46 and RP96-61 demonstrated the
presence of mutations in parC that had not been described previously. Isolate RP96-46 contained a mutation which resulted in an
amino acid change at position 116 from arginine to leucine. Isolate
RP96-61 contained not only the mutation at position Arg-116 but also an
additional mutation which caused an amino acid change at position 85 from glycine to cysteine.
This is the first report of the presence of double ParC mutations
identified in clinical isolates of N. gonorrhoeae. A double mutation was identified in a laboratory-induced resistant
strain(1); the locations of the ParC mutations identified in
this strain (Ser-88 and Gly-91) were different from the locations of
the double mutations that we describe here. Additional examination of
the three isolates containing the Ser-87Ile and Glu-91Gly ParC
mutations suggest that they are not separate isolations of the same
clone (Table 1). RP96-70 was auxotype/serovar class PA/IB-1 and
exhibited decreased susceptibility or resistance to penicillin and
tetracycline (CMRx), while RP96-c1-003 was PA/IB-32
(penicillinase-producing N. gonorrhoeae [PPNG]) and
RP96-c2-001 was PA/IB-1 (PPNG). The other two isolates in this study
present a different scenario. Isolates RP96-46 and RP96-61 belonged to
auxotype/serovar class Proto/IB-1 and were PPNG. RP96-46, for which the
ciprofloxacin MIC was 1.0 µg/ml, contained only the mutation
Arg-116Leu. In contrast, RP96-61 contained the Gly-85Cys mutation
in addition to the Arg-116Leu mutation, and the ciprofloxacin MIC
for this strain was 16.0 µg/ml. Acknowledging that transformation
studies need to be performed for confirmation, these results suggest
that the Arg-116Leu mutation alone may be sufficient to produce
clinically significant resistance (ciprofloxacin MIC, 1.0 µg/ml) but
that the addition of the Gly-85Cys mutation is necessary for the
high-level MICs for RP96-61 (ciprofloxacin MIC, 16.0 µg/ml).
In summary, we have identified five ciprofloxacin-resistant isolates of
N. gonorrhoeae that contain novel mutation patterns in
parC, including two double mutations, the Ser-87Ile and
Glu-91Gly mutations and the Gly-85Cys and Arg-116Leu
mutations. Of these mutations, the Gly-85Cys and Arg-116Leu
mutations in parC have not been previously observed in the
gonococcus. The importance of the double parC mutations
appears to be the high-level ciprofloxacin resistance exhibited by the
isolates which contain them. All four isolates which possess
parC double mutations have high-level resistance to
ciprofloxacin (MICs, 8.0 to 64.0 µg/ml), whereas for the isolate with
a single parC mutation the ciprofloxacin MIC was 1.0 µg/ml. That the three strains that contain the Ser-87Ile and
Glu-91Gly double mutations have different levels of ciprofloxacin
resistance is not unexpected. For strains which contain the same
mutation pattern with a single ParC mutation, a similar wide, although lower, range of MICs has been demonstrated (unpublished data). We
speculate that the presence of double ParC mutations facilitates the
high ciprofloxacin MICs for a strain, but the MIC may also be
influenced by other characteristics of the strain.
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ACKNOWLEDGMENTS |
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This study was supported in part by Emerging Infectious Disease funding from the National Center for Infectious Diseases, CDC. This study was supported in part by an appointment (A.L.S.) to the Research Participation Program at CDC administered by the Oak Ridge Institute for Science and Education.
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FOOTNOTES |
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* Corresponding author. Mailing address: Bacterial STD Branch, Mailstop G-39, Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333. Phone: (404) 639-2134. Fax: (404) 639-3976. E-mail: dlt1{at}cdc.gov.
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Antimicrobial Agents and Chemotherapy, August 1998, p. 2103-2105, Vol. 42, No. 8
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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