Fluoroquinolone resistance among Neisseria gonorrhoeae isolates from Shanghai, China: Detection of quinolone resistance-determining region mutations.
Methods: Eighty N. gonorrhoeae isolates were collected from Shanghai Skin Disease & Sexually Transmitted Disease Hospital or DongFang Hospital during April 2005 to April 2006 in Shanghai, P.R. of China. The minimum inhibitory concentrations (MIC) of fluoroquinolones for these isolates were determined by an agar dilution method. Mutation patterns within gyrA and parC were determined by direct sequencing or by using established restriction fragment length polymorphisms (RFLP) methods.
Results: Ninety five per cent (76 of 80) of isolates were resistant, 3.75 per cent (3 of 80) intermediate resistant, and 1.25 per cent (1 of 80) were sensitive to fluoroquinolone drug ciprofloxacin. Sequencing and RFLP analysis of gyrA and parC revealed that all resistant isolates had dual mutations of S91F and D95A/G/N in gyrA. Some isolates had an extra mutation within parC either of D86N, $87N or E91A/G. Mutation patterns for gyrA and parC were significantly (P<0.05) associated with MICs level.
Interpretation & conclusions: Mutations of S91F and D95A/G/N in gyrA combined with $87N in parC was the most prevalent mutation pattern of fluoroquinolone resistant N. gonorrhoeae isolates. This mutation pattern was associated with a high level of quinolone resistance (MIC >16.0 [micro]g/ml) which can serve as a maker for quinolone-resistance prediction in Shanghai, P.R. of China.
Key words Fluoroquinolone--gyrA--Neisseria gonorrhoeae--parC--resistance
Gonorrhoea is a sexually transmitted disease due to infection by bacterium Neisseria gonorrhoeae. Patients are usually treated with antibiotics. In China, antibiotic-resistant N. gonorrhoeae isolates are common due to irregular prescription of antibiotics, and have become an important public health concern (1,2).
Fluoroquinolones are frequently used in treatment for gonorrhoea, with ciprofloxacin and ofloxacin being used as primary drugs in a number of countries. N. gonorrhoeae was highly susceptible to ciprofloxacin when this drug was first introduced in 1980s. In the last few years, a number of gonococci with decreased susceptibility or clinically significant resistance to fluoroquinolones including ciprofloxacin have been isolated all over the world (3-8).
The mechanism of fluoroquinolone-resistance of N. gonorrhoeae has been a subject of investigation (9,10). One possibility is that the gonococcus involves mutations in the quinolone-resistance-determining-region (QRDR) of gyrA and the analogous of parC locus on the chromosome. These mutations result in altered GyrA and ParC proteins (9,11,12). Altered proteins can no longer be bound by fluoroquinolones, therefore, the drug is unable to inhibit DNA replication and bacterium becomes less susceptible. The level of drug susceptibility appears to correlate with the location and number of mutations presented (12). This mechanism is analogous to those observed in Escherichia coli or other bacterial This study was carried out to provide further evidence of the correlation of the pattern of mutation of gyrA and parC genes with the drug resistant of N. gonorrhoeae in isolates from Shanghai, P.R.China, where higher resistance to ciprofloxacin has been reported than those from other regions of the world (6).
Material & Methods
Bacterial isolates: N. gonorrhoeae isolates were obtained from urethral and endocervical swabs, taken from gonorrhoea patients who had visited Shanghai Skin Disease & Sexually Transmitted Disease Hospital or DongFang Hospital during the period of April 2005 to April 2006. A total of 435 patients visited these hospitals for sexually transmitted disease treatment during the study period. Case exclusion criteria included: (i) co-infection with pathogens other than N. gonorrhoeae; and (ii) antibiotic treatment for the current episode of infection before enrollment. All eligible patients gave written consents for their enrollment. A total of 80 isolates were successfully obtained from patients who met the inclusion requirements of this study. Reference strains WHO-A, B, C, D and E were provided by National Institute for Control of Pharmaceuticals and Biological Products (Beijing, P.R. of China).
Antibiotics: Ofloxacin (Daiichi Sankyo Ltd, Tokyo, Japan), lomefloxacin (Abbott Ltd, Illinois, United States) and ciprofloxacin (Bayer Ltd, Leverkusen, Germany) were provided by National Institute for Control of Pharmaceuticals and Biological Products (Beijing, P.R. of China).
Minimum inhibitory concentration (MIC): An agar dilution method recommended by the WHO Western Pacific Regional Resistance Surveillance Programme was used to determine minimum inhibitory concentration (MIC) (13). MIC tests were performed on chocolate agar base (Oxiod Ltd, Basingstoke, United Kingdom) supplemented with 10 per cent defibrinated fresh sheep blood (Zhongqing Biotech Inc. Ltd, Shanghai, China) and 1 per cent Iso VitaleX (Oxiod Ltd, Basingstoke, United Kingdom). Agar plates were inoculated with [10.sup.8] cfu/ml bacteria, incubated at 36[degrees]C with 5 per cent C[O.sub.2] for 36 h. Antibiotics were diluted in agar at concentrations from 0.002 to 16.0 [micro]g/ml for ciprofloxacin, 0.0078 to 16.0 [micro]g/ml for ofloxacin and 0.0078 to 16.0 [micro]g/ml for lomefloxacin. Reference strains (WHO A-E) with known MICs were co-tested with samples as control. MICs were determined as the lowest antibiotic concentration that had completely inhibited bacteria growth (14).
Amplification of quinolone-resistance-determining-region: Oligo primers were synthesized by Sangon Biotech Co, Ltd. (Shanghai, P.R. of China). Primers for amplification of QRDR within gyrA were: Forward 5'-CGC GAT GCA CGA GCT GAA AAA-3', Reverse 5'-ATT TCG GTA TAG CGC ATG GCT G-3'; for QRDR within parC were: Forward 5'-GTT TCA GAC GGC CAA AAG CCC-3', Reverse 5'-GGA CAA CAG CAA TTC CGC AAT-3'. PCRs were carried on using 25 [micro]l volume that consisted of 12.5 [micro]l 2XPCR master mix (Fermentas UAB, Vilnius, Lithuania), 9.5 [micro]l sterile water, 1 [micro]l each of forward and reverse primers (0.2 [micro]M) and 1 [micro]l DNA template. Reaction condition was 35 cycles of denaturation for 60 sec at 94[degrees]C, annealing for 50 sec at 52[degrees]C, and extension for 50 sec at 72[degrees]C.
Sequencing of gyrA and parC: PCR products were separated by 1.5 per cent agarose gel electrophoresis, purified by PCR purification Kit (QIAGEN, Hilden, Germany) and sequenced by Bigdye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, United States). Mutations were identified by comparing translated amino acid sequences to reference gyrA sequence (Genbank accession no: U08817) or parC sequence (Genbank accession no: U08907).
Analysis of restriction fragment length polymorphism: Restriction fragment length polymorphism (RFLP) analysis was carded out as described earlier (15,16). Primers used for gyrA PCR amplification were: gyrA-F 5'-CGC GAT GCA CGA GCT GAA AAA-3' and gyrA-HinfI 5'-CCG TCT ATC AGC ACA TAA CGC ATA GCG AAA TTT TGC GCC ATA CGG ACG ATG GAG-3' Amplicon was further digested with endonuclease Hinf I (Fermentas UAB, Vilnius, Lithuania) for detection of mutations at nucleotides 272 and 284. Primers used for parC PCR amplification were: parCF 5'-AAG CCG GTG AAA TCG GCG CGC-3', paired with ParC-Sal I 5'-GAG AAT TTG GGT AAA TAC CAT CCG CAC GTC-3', ParC-Pst I 5'-GGT AAA TAC CAT CCG CAC GGC TGC-3', ParC-Hinf I 5'-AAT CCT GAG CCA TGC GCA CCA TCG AC-3' or parCR 5'-GTC GCC GTC GCG CGA ACC GAA-3'. Amplicons were each digested with Sal I, Pst I or Hinf I (Fermentas UAB, Vilnius, Lithuania) for detection of mutations at nucleotides 256, 260 and 272 of parC gene.
Statistical analysis: Association of mutation patterns with fluoroquinolone resistance was examined by Mann-Whitney test and Kruskal-Wallis test. Tests were performed using Statistic Packages for Social Science 10.0 (SPSS Inc., Chicago, IL, United States).
Resistance to ciprofloxacin, ofloxacin, and lomefloxacin were observed in 95.0 (76 of 80), 95.0 (76 of 80) and 97.5 (78 of 80) isolates respectively (Table I). One isolate was susceptible to ciprofloxacin with an MIC less than 0.06 [micro]g/ml.
Together with WHO-A, 14 isolates were randomly selected for a preliminary sequencing analysis. One sensitive isolate (MIC=0.03 [micro]g/ml), two intermediated resistant isolates (MIC >0.125 [micro]g/ml and <0.5 [micro]g/ ml) and 11 resistant (MIC=1.0-16.0 [micro]g/ml) were included. All intermediate and resistant isolates had a common mutation S91F in gyrA. Twelve out of 13 intermediate and resistant isolates had a mutation at codon 95 of gyrA, among these there were mutations D95G (5 isolates), D95A (6 isolates) and D95N (one isolate). The other isolate R302 demonstrated a mutation A92P, which was seldom reported in the mainland China. Sequence analysis of parC showed a variety of mutations at codons 86, 87 and 91, while some synonymous mutations were detected in other positions (Table II). Result of 14 selected isolates, analyzed with both sequencing and RFLP method were consistent with each other (Table II). It indicated that RFLP methods could be used to detect mutations in the QRDR in the gyrA and parC, except for the A92 mutation, a seldom reported mutation site.
Since pilot sequence analysis had shown that mutations of gyrA and parC mostly took place at gyrA 91 and 95, parC 86, 87 and 91 codons, so all other isolates were re-examined by using RFLP. Theoretically, a wide type of gyrA gene amplified with the primers gyrA-F and gyrA-Hinf I could yield a 165bp product, which contains a natural Hinf I cleavage site at Scr 91 codon and an artificially created cleavage site at Asp 95 codon. Consequently, Hinf I could digest the amplified fragment to produce three products with lengths of 96 bp, 54 bp and 15 bp, respectively. When this region had mutations, Hinf I cleavage cite at Scr 91 and/or Asp 95 would be destroyed. Therefore, digestion of PCR products with Hinf I would produce restriction fragment length polymorphism to show the mutations of gyrA gene. Representative data from the experiments are shown in Fig. a. Digestion of the wild type WHO-A resulted in 96 bp and 54 bp fragements. Isolate R302 (lane 1) yielded 111bp and 45 bp fragments, indicating only a mutation at codon 91 was detected, while a mutation at Ala 92 could not be detected. All other isolates yielded a 165 bp fragement, indicated mutations at both codon 91 and 95. In other words, All isolates, including the sensitive isolate, had mutations in the gyrA gene. All isolates resistant or intermediately resistant to fluoroquinolones had a mutation at Ser 91, and a mutation at Asp 95 or Ala 92.
Three DNA fragments can be amplified from parC gene with primer pairs of parC-sal I/parCR, parC-Pst I/parCR or parCF/parC-Hinf I. Lengths of each fragment were 132 bp, 123 bp and 105 bp, respectively. When these isolates had any mutations at codons of parC 86, 87 or 91, such as D86N, S87N/D or E91A/G/R91, the cleavage sites would change and the restriction enzyme digested fragments could show some polymorphisms in length. Two isolates (lane 12 and 15) (Fig. b) could not be digested by sal I, indicating a mutation at Asp 86. Six isolates (lane 21, 24, 27, 28, 29, and 30) (Fig. c) could not be digested by Pst I, indicating a mutation at Ser 87. Three isolates (lane 32, 33, and 37) (Fig. d) could not be digested by Hinf I, indicating a mutation at Glu 91. Overall, 12 isolates had a mutation at parC86, 44 isolates had a mutation at parC87, 7 isolates had a mutation at parC91.
RFLP results were summarized in Table III. It had shown MICs and mutations of gyrA and parC for all resistant isolates in this study. A significant association (P<0.05) was observed between mutation patterns with the level of MICs. Isolates with mutations in gyrA combined with parC87 mutation showed a significantly (P<0.01) higher level of resistance to ciprofloxacin (MIC > 16.0 [micro]g/ml, 31.8 per cent (14 of 44)) than just with gyrA only mutations.
In the last decade, the third generation cephalosporin and fluoroquinolones were recommended for the treatment of gonococeal infections worldwide. In recent years, there have been many reports on increasing number of quinolone resistant strains in Untied States, as well as in other countries (6-8,17-20), which has compromised its utility. According to the WHO Gonococcal Antimicrobial Surveillance Programme (GASP) results, the proportion of resistance was less than 16 per cent before 1995. After 1997, quinolone resistance had been increased rapidly and the resistance proportion reached to 80 per cent. It had been reported as high as 94.3 per cent in 2003 (21-22). Our results (96% resistant isolates) also showed that fluoroquinolones are no longer suitable for clinical treatment of gonorrhoea in Shanghai.
Quinolones have a bactericidal effect when these bind with two target enzymes, DNA gyrase and topoisomerase IV, which are essential for DNA replication within the cell. The lethal effect of quinolone occurs when an intermediary complex of drug and enzymes blocks its replication, and gyrA and parC genes encode two key target enzymes. The important mechanism for the fluoroquinolone resistance in the gonococcus involves mutations in the analogous region of QRDR of gyrA and parC in Escherichia coli, which was first reported by Belland et al (9). The pattern of mutations within gyrA and parC varied in different regions. Mutations at gyrA codon 91 and 95 were more often reported than mutation at gyrA codon 92. Compared with gyrA, mutations within parC were more variable All over the world. Mutations at parC codon 86, 87, and 91 were often reported (23). An earlier study in mainland China had shown that the main mutations were S91F and D95G in gyrA, S87N and D86N in parC (24). A study conducted in Hong Kong had shown that the most prevalent mutation pattern was S91F and D95G in gyrA and S87R in parC, which accounted for about 25 per cent of total mutations; the second most frequent pattern was S91F and D95G in gyrA, which accounted for 21 per cent (25). In this study, the major mutation pattern was S91F and D95G/A in gyrA and S87R/N in parC. Mutations in gyrA that lead to substitutions of phenylalanine for serine at position 91 were consistent in All isolates in this study, while Asp95 had more mutation patterns that could also be detected in the sensitive strain. Results from this study and other studies in China had suggested that Ser91 mutation might play an important role in mediating quinolone resistance in gonococci (24,26-27). DNA gyrase had two subunits, GyrA and GyrB, which were encoded by gyrA and gyrB, while topoisomerase IV was encoded by parC and parE genes (28). It may be a primary target of fluoroquinolones. Mutations in parC could be detected simultaneously with a mutation in gyrA, suggesting that this mutation may play a compensatory role in the resistance mechanism. Most mutation patterns, which had been reported in other countries, have also been detected in Shanghai (10,23,29).
RFLP analysis had indicated that All intermediate and resistant isolates had mutations in QRDR of gyrA and/or parC. The resistance level of isolates with a mutation in gyrA combined with parC was higher than that in those isolates with only gyrA mutation. It suggests that mutations in gyrA might determine the main resistance ability to quinolone, while additional parC mutation might mediate a higher level of resistance to quinolones.
In conclusion, mutation patterns revealed in this study, showed that S91F in gyrA could serve as a quinolone resistance marker for isolates from Shanghai, while S87R/N in parC could serve as a high-level quinolone resistance marker.
Received July 20, 2007
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Reprint requests: Dr Zhou Xiaoming, Department of Epidemiology, Shanghai Public Health Clinical Centre CaoLang Road 2901, Shanghai 201508, P.R. of China e-mail: firstname.lastname@example.org, email@example.com
Zhang Tiejun, Zhou Xiaoming , Zhang Jilun , Zhang Yinghu , Ren Yanhua , Chen Yue , Gu Weiming , Zhang Tao & Jiang Qingwu
Department of Epidemiology, School of Public Health, Fudan University, PR of China; Key Laboratory of Public Health Safety, Ministry of Education;  Shanghai Public Health Clinical Centre, Fudan University Affiliated, PR of China,  Shanghai Entry-Exit Inspection & Quarantine Bureau;  Shanghai Minghang Centre for Disease Control & Prevention;  Shanghai Pudong Centre for Disease Control & Prevention, PR of China;  Department of Epidemiology & Community Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Canada;  Shanghai Skin Disease & Sexually Transmitted Disease Hospital, PR China
Table 1. Minimum inhibitory concentration (MIC) of isolates (n=80) for ciprofloxacin, ofloxacin and lomefloxacin Antibiotics Distribution of MIC ([micro]g/ml) 0.0078 0.0156 0.0312 0.0625 0.125 0.25 Ciprolloxacin 0 0 1 0 0 -1 Ofloxacin 0 0 0 0 0 1 Lomefloxacin 0 0 0 0 1 0 Distribution of MIC ([micro]g/ml) 0.5 1.0 2.0 4.0 8.0 16.0 32.0 64.0 Ciprolloxacin 3 5 44 8 5 14 0 0 Ofloxacin 2 1 14 43 19 0 0 0 Lomefloxacin 0 1 78 0 0 0 0 0 Antibiotics Distribution of MIC ([micro]g/ml) [MIC.sub.50] [MIC.sub.90] 128.0 ([micro]g/ml) ([micro]g/ml) Ciprolloxacin 0 2.0 16.0 Ofloxacin 0 4.0 8.0 Lomefloxacin 0 2.0 2.0 Table II. Mutations in gvrA and parC revealed by both preliminary sequencing analysis and RFLP analysis Sequencing results Isolates MIC ([micro] gyrA g/ml) 91 92 95 WHO-A 0.016 R345 0.03 Asp to Ala D347 0.5 Ser to Phe Asp to Gly R307 0.5 Ser to Phe Asp to Gly D327 1 Ser to Phe Asp to Asn D339 1 Ser to Phe Asp to Ala G313 1 Ser to Phe Asp to Ala R302 2 Ser to Phe Ala to Pro D301 2 Ser to Phe Asp to Gly D356 2 Ser to Phe Asp to Ala 8341 4 Ser to Phe Asp to Ala Q317 4 Ser to Phe Asp to Gly R306 8 Ser to Phe Asp to Ala R335 8 Ser to Phe Asp to Ala R316 16 Ser to Phe Asp to Gly Sequencing results Isolates parC 86 87 91 WHO-A R345 D347 R307 Ser to Asn D327 D339 G313 Glu to Ala R302 D301 D356 8341 Asp to Asn Q317 Glu to Gly R306 Glu to Ala R335 R316 Ser to Arg RFLP results Isolates gyr4 parC Mutation site Mutation site WHO-A None None R345 Asp95 None D347 Ser-91, Asp95 None R307 Ser91, Asp95 Ser87 D327 Ser91, Asp95 None D339 Ser91, Asp95 None G313 Ser91, Asp95 Glu91 R302 Ser91 * None D301 Ser91, Asp95 None D356 Se r91, Asp95 None 8341 Set-9 1, Asp95 Asp86 Q317 Ser91, Asp95 Glu91 R306 Ser91, Asp95 Glu91 R335 Set-9 1, Asp95 None R316 Ser91, Asp95 Ser87 MIC: value for ciprofloxacin; MIC [less than or equal to] 0.06, sensitive; MIC 0.125~0.5, intermediate resistant; MIC [greater than or equal to] 1.0, resistant-. Ala92 could not be detected here, because of no endonuclease recognized cleavage site Table III. Minimum inhibitory concentrations (MIC) and mutation patterns for isolates (n-80) Mutations MIC ([micro] g/ml) 0.5 1.0 2.0 4.0 8.0 16.0 total gvrA 2 3 10 2 2 19 gvrA+parC86 9 1 1 1 12 gvrA-parC87 1 1 23 4 1 14 44 gvrA+parC91 1 3 1 1 1 7 MIC, value for ciprofloxacin Some isolates had both mutations in parCS6 and parCS7, so they had been calculated twice in this Table. The sensitive isolate was not included