1 Department of Laboratory Medicine, 2 Research Institute of Bacterial Resistance and 3 Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul; 4 Department of Bacteriology, National Institute of Health, Seoul, Korea; 5 WHO Collaborating Centre for STD and HIV, Department of Microbiology, The Prince of Wales Hospital, Sydney, Australia
Received 7 March 2004; returned 24 April 2004; revised 22 May 2004; accepted 2 June 2004
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Abstract |
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Methods: The antimicrobial susceptibilities of 190 isolates of gonococci from Korea in 2000 were examined by NCCLS methods, and subsets of these isolates underwent mutation analysis of the quinolone resistance-determining regions (QRDRs) of gyrA and parC. Molecular epidemiological characterization of 25 Korean isolates and 54 isolates from overseas was performed by pulsed-field gel electrophoresis (PFGE) and the results compared.
Results: Most (172, 90.5%) of the 190 gonococci tested displayed reduced susceptibility to ciprofloxacin. All strains with high-level ciprofloxacin resistance (ciprofloxacin MIC 4 mg/L) contained a double amino acid alteration at the 91 and 95 positions in the QRDR of GyrA and a single alteration in ParC. PFGE types of high-level QRNG in Korea were mostly different from those of other nearby countries.
Conclusions: These results suggest that the observed increase in ciprofloxacin-resistant isolates is due to the mutation and spread of Korean multiclonal isolates rather than importation from overseas.
Keywords: N. gonorrhoeae , pulsed-field gel electrophoresis , ciprofloxacin , resistance , QRDRs
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Introduction |
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In recent years, a decrease in the proportion of penicillinase-producing N. gonorrhoeae (PPNG) and a rapid increase in quinolone-resistant N. gonorrhoeae (QRNG) have been reported to be related in some countries.35 However, in Korea, the proportion of PPNG has remained high (84%), and strains with reduced susceptibility to ciprofloxacin increased rapidly from 9% in 1992 to 84% in 1999.6,7 This phenomenon may be caused by the spread of strains in which the gyrA and parC mutations arose locally or by the importation of QRNG from overseas followed by sustained domestic transmission of resistant subtypes. In either case, the QRNG may be of multiple subtypes or else represent the expansion of limited numbers of subtypes.
The aims of this study were to examine the cause of the increase in QRNG observed in Korea by testing antimicrobial susceptibility, analysing the mutations of the quinolone resistance-determining regions (QRDRs) of gyrA and parC of fluoroquinolone-non-susceptible isolates, and comparing the molecular epidemiological characteristics of QRNG in Korea with those of other nearby countries.
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Materials and methods |
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One hundred and ninety gonococci were isolated from Korean patients or prostitutes attending sexually transmitted diseases clinics in Seoul in 2000 using modified Thayer-Martin agar (BBL, Becton Dickinson, Cockeysville, MD, USA). Species identification was based on conventional culture and biochemical characteristics.8 The isolates were kept frozen at 70°C until used.
For comparison purposes, 54 random gonococcal isolates from patients infected in nearby countries, namely, Australia, China, Mongolia, the Philippines, India, Malaysia, Singapore, Thailand, Vanuatu and Vietnam from 1995 to 2000, were obtained from the regional World Health Organization (WHO) reference laboratory in Sydney, Australia.
Antimicrobial susceptibility testing
The medium used for susceptibility testing was GC II agar base supplemented with 1% IsoVitaleX (Becton Dickinson). All 190 Korean isolates were initially examined by the disc diffusion testing methods of the NCCLS9 using a 5 µg ciprofloxacin disc (Becton Dickinson).
Forty-nine Korean gonococcal strains isolated consecutively between April and June 2000, and the 54 gonococci from nearby countries were examined for susceptibility by the NCCLS agar dilution method.10 Seventeen of 20 isolates with no ciprofloxacin inhibition zone among 190 isolates were tested by the agar dilution test to determine the level of ciprofloxacin resistance. Ciprofloxacin powder was supplied by Bayer Korea (Seoul, Korea). Inocula of 104 cfu were applied using a Steers replicator (Craft Machine, Chester, PA, USA). Plates were incubated in a 5% CO2 incubator at 35°C for 24 h after which the results were read. Strains with high-level ciprofloxacin resistance were defined as those for which the MIC was
4 mg/L.11 N. gonorrhoeae ATCC 49226 was used as a control strain. During 2000 and 2001, our laboratory accurately categorized those QRNG included in an external quality assurance programme conducted by the WHO Western Pacific Region Gonococcal Antimicrobial Surveillance Programme (GASP).12
Sequencing the QRDRs of gyrA and parC
Twenty-nine Korean gonococcal isolates with various ciprofloxacin disc susceptibility patterns were randomly selected, and the ciprofloxacin MICs for these isolates were also determined by the agar dilution method. The QRDRs of these isolates were investigated by sequencing and these results were correlated with the MICs of ciprofloxacin. The QRDR sequences of the gyrA and parC genes were amplified using PCR primers as reported by Tanaka et al.3 PCR was carried out with 1 µL of heat-extracted template DNA, 10 pmol of each primer and PreMix (Bioneer, Cheongwon, Korea) containing 1 U of Taq DNA polymerase in a total volume of 20 µL. A thermal cycler (Eppendorf, Hamburg, Germany) was used under the following conditions: 35 cycles of 93°C for 30 s, 58°C for 30 s and 72°C for 1 min. The PCR products were extracted using a DNA extraction kit (Qiagen, Hilden, Germany), and their nucleotide sequences were determined by the dideoxynucleotide-chain termination method with ABI PRISM BigDye Terminator Sequencing Kits (Perkin-Elmer, Foster City, CA, USA) and using an automatic DNA sequencer (ABI 3700; Perkin-Elmer).
Epidemiological study
Pulsed-field gel electrophoresis (PFGE) was performed on a total of 79 strains for which the ciprofloxacin MICs were determined by agar dilution methods, namely, 25 of the 29 randomly selected isolates from Korea, and the 54 gonococci from overseas, as described previously.7 Briefly, the plugs were prepared by using saline EDTA solution-suspended cells cultured overnight. The genomic DNA was digested with NheI (Takara, Tokyo, Japan) for 18 h at 35°C. The CHEF DR II instrument (Bio-Rad, Hercules, CA, USA) was used to separate the fragments, with switch times of 0.5 s (initial) and 54 s (final), and a running time of 20 h at 6 V/cm. PFGE banding pattern similarities were determined using the Dice coefficients and unweighted pair group method using arithmetic averages (UPGMA) clustering method using Molecular Analyst Fingerprinting Software (v. 1.12; Bio-Rad). A tolerance in the band positions of 0.5% was used. An 80% similarity threshold was used to divide the outputs from the dendrogram into clusters.
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Results |
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Ciprofloxacin disc susceptibilities of the 190 isolates were: 18 (9.5%) susceptible, 122 (64.2%) intermediate and 50 (26.3%) resistant. For the 17 isolates with no ciprofloxacin inhibition zone, ciprofloxacin MICs were 16 mg/L (data not shown). The susceptibilities of the 49 Korean and the 54 non-Korean isolates, as determined by agar dilution methods, are shown in Table 1. The MIC range of ciprofloxacin for N. gonorrhoeae was
0.01532 mg/L, and the geometric mean ciprofloxacin MIC was 0.396 mg/L. The ciprofloxacin susceptibility categories of the 54 overseas isolates were: eight susceptible, 12 intermediate and 34 resistant. Among these isolates, 22 (40.7%) were high-level ciprofloxacin resistant with MICs of
4 mg/L.
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The ciprofloxacin MICs for 29 randomly selected Korean isolates are shown in Table 2 together with details of the QRDR alterations present. All 17 of the high-level ciprofloxacin-resistant Korean isolates contained three amino acid substitutions in the GyrA and ParC proteins. Of these, the most common substitutions (in 16 of 17 strains) were serine to phenylalanine at position 91 and aspartic acid to glycine at position 95 in GyrA, and serine to arginine at position 87 in ParC. The isolates for which ciprofloxacin MICs were 0.122 mg/L contained one or two amino acid substitutions in GyrA alone. No alterations in GyrA and ParC were detected in the three susceptible strains.
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Among the 25 Korean isolates examined, 13 PFGE types were observed: eight strains (32.0%) were E type and four (16.0%) type (Figure 1). In contrast, 33 PFGE types were observed among the 54 overseas isolates: five strains (9.3%) were Q type and four (7.4%) showed ß and
types.
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Discussion |
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Generally, gyrA mutations play an important role in the development of fluoroquinolone resistance in gonococci, and simultaneous parC mutations play a complementary role in increasing the level of resistance.14,15 Shultz et al.11 also reported that the sequence and nature of the QRDR changes were found to correlate with MICs of ciprofloxacin, and some parC alterations do not have a significant effect on MIC. In this study, we found that all strains with high-level ciprofloxacin-resistance contained a double amino acid alteration at the 91 and 95 positions in the QRDR of GyrA and a single alteration in ParC. These findings concur with those of other investigators.14,15 However, the amino acid substitutions in Korean isolates with high-level ciprofloxacin resistance were of a similar configuration in 16 of 17 instances, suggesting that clonal expansion of a QRNG may have been involved. The amino acid substitutions in the predominant QRDR change seen differed from those reported by some other investigators in the region,3,13,15 although they were also observed in a QRNG isolated from an infection acquired in China.11
One of the aims of this study was to determine whether the increase in high-level ciprofloxacin-resistant N. gonorrhoeae observed in Korea was caused by the importation of resistant strains from overseas, or by the spread of resistant variants, perhaps selected by antibiotic pressures in Korea.
N. gonorrhoeae strains are genetically diverse due to the frequent transfer and recombination of genetic elements between strains.16 Su & Lind15 reported that a common epidemic clone might be widely spread in the Philippines, Hong Kong, Romania, New Guinea and Denmark, but the study was based on examination of small numbers of ciprofloxacin-resistant isolates. In contrast, Trees et al.13 reported that ciprofloxacin-resistant isolates in Bangkok were not from a single clonal source, but of diverse subtypes from multiple cases of importation or local emergence.
Among the 25 Korean isolates examined, 13 PFGE types were found (Figure 1). Of these, 13 isolates were high-level ciprofloxacin resistant and all had the same QRDR changes. Eight of the 13 high-level ciprofloxacin-resistant strains were of the E type, indicating a significant degree of strain relatedness. However, the other five high-level resistant strains with the same QRDR alterations were from unrelated PFGE types (four and one W2). One high-level ciprofloxacin-resistant W2-type Korean isolate was closely related to a ciprofloxacin-susceptible W1-type Korean isolate. The PFGE patterns of the high-level ciprofloxacin-resistant strains from Korea differed from those of isolates in other nearby countries. It is tempting to suggest from these results that the observed increase in high-level ciprofloxacin-resistant strains in Korea may have arisen as a result of the spread of multiclonal resistant variants of domestic strains. In this circumstance, the predominant QRDR change observed could have arisen locally and spread amongst already circulating strains. This phenomenon has been observed in a study of a closed population in Mongolia.17 Among the isolates with K,
2 and
types in this study, all Korean isolates were ciprofloxacin-susceptible, while some non-Korean isolates were ciprofloxacin-resistant, suggesting that the resistance was not introduced from overseas.
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Acknowledgements |
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Footnotes |
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References |
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