J Med Microbiol 55 (2006), 1251-1255; DOI: 10.1099/jmm.0.46401-0
© 2006 Society for General Microbiology
ISSN 1473-5644
Antimicrobial susceptibility of Neisseria gonorrhoeae isolated in Jiangsu Province, China, with a focus on fluoroquinolone resistance
Bei Wang1,
Jin-shui Xu1,
Chang-xian Wang1,
Zu-huang Mi2,
Yue-pu Pu1,
Mamie Hui3,
Thomas K. W. Ling3 and
Chiu-Yeung Chan3
1 School of Public Health, Southeast University, Nanjing, Jiangsu, People's Republic of China
2 Wuxi Clon-Gen Technique Institute, Wuxi, Jiangsu, People's Republic of China
3 Department of Microbiology, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong Special Adminstrative Region, People's Republic of China
Correspondence
Yue-pu Pu
puyuepu{at}yahoo.com.cn
Received 5 November 2005
Accepted 1 June 2006
In this study, the phenotypic and genotypic resistance to fluoroquinolones in Neisseria gonorrhoeae isolated in Jiangsu Province, China, was analysed. In vitro susceptibility testing of eight antimicrobial agents, including ciprofloxacin and levofloxacin, against 95 clinical isolates was carried out. Detection of mutations in the gyrA and parC genes was performed by sequence analysis. The clinical isolates demonstrated 100 % resistance to ciprofloxacin and 98.9 % non-susceptibility to levofloxacin. All of the isolates were susceptible to cefotaxime and ceftriaxone. For cefepime, spectinomycin and tetracycline, 98.9, 94.7 and 1.1 % of the isolates were susceptible, respectively. None of the isolates was susceptible to penicillin. Five types based on gyrA mutations could be categorized among 54 isolates with seven different mutation sites found on their parC gene. Analysis of sequence results showed that the gyrA mutation Asp-95
Ala and the parC mutations Ser-87Arg and Ser-87Asn made a significant contribution to the resistance to fluoroquinolones, in addition to double mutations found in each gene. Therefore, the use of fluoroquinolones in the treatment of N. gonorrhoeae infections in Jiangsu Province is not recommended, while the use of third- and fourth-generation cephalosporins and spectinomycin is recommended.
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INTRODUCTION
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Neisseria gonorrhoeae is a major pathogen of sexually transmitted diseases in China, with a cumulative total of 185 319 notified cases in 2003 (Chinese Centre for Disease Control and Prevention, 2004). Nowadays, treatment of gonorrhoea using chloramphenicol, sulphonamide, penicillin and tetracycline has become ineffective. The fluoroquinolones were introduced and widely used clinically in the 1980s because of their effectiveness and convenience in single-dose oral administration, together with lower cost. After 10 years of clinical application, an increasing trend of fluoroquinolone resistance in N. gonorrhoeae has been reported in Japan, Korea, south-east Asia, Canada and China (Krishna et al., 2005; Ng et al., 2002; Tanaka et al., 2004; World Health Organization, 2003; Yoo et al., 2004). In China, the rate of fluoroquinolone resistance is about 40–80 %, depending on the region studied (Gu et al., 2004; Ye, 2001). However, in China, quinolones are still recommended to treat gonorrhoea (single dose 500 mg for ciprofloxacin) (Cai et al., 2003; Su, 2002; Su et al., 2004; World Health Organization, 2003; Zhu & Zhao, 2003). Accordingly, it is necessary to investigate the resistance rate and mechanism in N. gonorrhoeae. It has been reported that the development of mutations in DNA gyrase subunit A (GyrA) encoded by the gyrA gene and mutations in topoisomerase IV encoded by the parC gene account for most ciprofloxacin resistance (Belland et al., 1994). We collected 95 isolates of N. gonorrhoeae from three regions of Jiangsu Province during 2003 to study their susceptibilities to eight antimicrobial agents and assess the correlation between fluoroquinolone MICs and amino acid changes in GyrA and ParC.
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METHODS
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Bacterial isolates and culture.
Ninety-five non-duplicate isolates of N. gonorrhoeae were collected from patients who attended sexually transmitted disease (STD) clinics in the Nanjing, Xuzhou and Wuxi regions between May and December 2003. The organisms were cultured and isolated on NG-Thayer-Martin agar (bioMérieux), and identified by Gram stain and oxidase reaction, followed by carbohydrate-utilization studies of glucose, maltose, fructose and sucrose (API NH, bioMérieux). The bacterial isolates were kept on chocolate agar slants (bioMérieux) for short-term storage. Pure cultures supplemented with 8 % skimmed milk and 10 % fetal calf serum were used for long-term storage at –70 °C.
In vitro antimicrobial susceptibility testing.
Material of known potency of the following antimicrobial agents was used for MIC determination: ciprofloxacin (Bayer), levofloxacin (Daiichi), spectinomycin (Pharmacia and Upjohn), penicillin (Sigma), tetracycline (Sigma), cefotaxime (Sigma), cefepime (Bristol-Myers-Squibb) and ceftriaxone (Sigma). MIC values were determined by an agar dilution method with GC agar base (Oxoid) plus supplements according to the National Committee for Clinical Laboratory Standards (2002). A multipoint inoculator (MIC-2000 System, Dynatech) was used to deliver 104 c.f.u. per spot onto GC agar base supplemented with 1 % Vitox (Oxoid) and 5 % lysed horse blood. N. gonorrhoeae ATCC 49226 was included as the control strain for the antimicrobial susceptibility test. The MIC (mg l–1) was defined as the concentration at which no colony was detected after agar plates were incubated at 37 °C in 5 % CO2 for 24 h. Resistance breakpoints for the antimicrobial agents penicillin, cefotaxime, ceftriaxone, cefepime, tetracycline, ciprofloxacin and spectinomycin were interpreted according to the National Committee for Clinical Laboratory Standards (2002). For levofloxacin, MIC values of 
1 mg l–1 were considered to be resistant (Shigemura et al., 2004; Centers for Disease Control and Prevention, 2005).
Detection of mutations in gyrA and parC genes.
Bacterial isolates were selected from every ciprofloxacin MIC level by the use of a stratified random-sampling method. Bacterial cells were harvested and suspended in Tris/EDTA buffer. Cell suspension (100 µl) was centrifuged at 10 000 g at 4 °C for 5 min, the pellet was resuspended in lysing buffer (Tris/EDTA containing 200 ng proteinase K ml–1) at 55 °C for 1 h, and then incubated at 95 °C for 5 min, centrifuged at 10 000 g for 5 min, and the supernatant collected and used as template. Primers for the gyrA gene were: forward 5'-GTA CTG TAC GCG ATG CAC GA-3' and reverse 5'-CGA GCC GTT GAC GAG CAG T-3', leading to a product of 372 bp. The amplification kit was obtained from the Wuxi Clon-Gen Technique Institute with the following protocol: 94 °C denaturation for 2 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 60 s, with final extension at 72 °C for 2 min. Primers for amplification of the parC gene were: forward 5'-GCA CGC TTC CCA TAC CGA-3' and reverse 5'-TCC ACC GTC CCC TGA TTG-3', leading to a product of 410 bp. The amplification protocol for the parC gene was identical to that for the gyrA gene, with the exception of denaturation at 93 °C. The primers for gyrA and parC were designed according to primer design principles and compared with sequences of homologous genes in GenBank. The amplified fragment of gyrA is from position 175–546, that of parC from position 15–443. The PCR products were sent to Shanghai BioAsia Biotechnology for sequence analysis on an ABI377 automatic sequencer (Applied Biosystems). The PCR product sequences were aligned, analysed and compared with the sequence of a wild-type strain of N. gonorrhoeae (GenBank accession nos U08817 and U08907) by Bioedit software (Ibis Therapeutics).
Data analysis.
SAS 8.1 software (SAS Institute) was used for ANOVA analysis and multiple regression analysis of correlation between mutation sites and MIC results.
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RESULTS AND DISCUSSION
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The 95 non-duplicate N. gonorrhoeae isolates were collected from 78 males and 17 females, with a mean age of 33.96 years (SD 10.49 years).
The antimicrobial MIC distribution for 95 isolates of N. gonorrhoeae is shown in Table 1
. All of the isolates were susceptible to cefotaxime and ceftriaxone. One isolate was susceptible to tetracycline. None of the isolates was susceptible to penicillin. Spectinomycin resistance was very low: only one isolate showed an MIC >128 mg l–1. One isolate was resistant to cefepime with MIC=2 mg l–1. All isolates were considered resistant to ciprofloxacin and non-susceptible to levofloxacin (Table 1).
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Table 1. MIC summary and susceptibility for 95 N. gonorrhoeae isolates
Abbreviations: CFT, ceftriaxone; CIP, ciprofloxacin; CPE, cefepime; CTX, cefotaxime; LEV, levofloxacin; PEN, penicillin; SPC, spectinomycin; TET, tetracycline; S, sensitive; I, intermediate; R, resistant.
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Of the 95 isolates, 60 were selected according to the ciprofloxacin MIC distribution. However, two of these could not be amplified and four could not be sequenced. As a result, 54 isolates were selected for PCR of amino acid changes in the GyrA and ParC subunits. Five types of gyrA mutations could be categorized, with amino acid changes mainly found in positions 91 and 95. Eleven mutation sites were found on the parC gene, leading to seven amino acid changes (Table 2), Four silent mutations were also detected (bases 312TC, 387GA, 393CG and 414GA) that gave no amino acid change.
Statistical analysis of the relationships between different mutations in the gyrA and parC genes, and levels of fluoroquinolone resistance, revealed significant correlations. It was noted that when more mutations were found in the two genes of a particular isolate, the organism became more resistant, with a higher MIC, as found with other bacterial species (Hawkey, 2003). Isolates with only gyrA mutations had lower MICs than those with mutations in both gyrA and parC (P<0.05 for ciprofloxacin; Table 3). In addition, analysis also revealed that mutations in gyrA resulting in Asp-95Ala and in parC resulting in Ser-87Arg and Ser-87Asn had significant influences on the elevation of MIC values (P<0.05).
Fluoroquinolones have been widely used to treat infections caused by N. gonorrhoeae since the 1980s because of their strong antibacterial activities, comparatively low costs and convenience of oral administration. However, resistance to these drugs emerged and spread quickly in the 1990s (Gu et al., 2004; Ye, 2001). Resistance to ciprofloxacin from N. gonorrhoeae isolated in Nanjing has increased from 2.89 % in 1994 to 97.17 % in 2002 (Su et al., 2004). Our results in this study indicated that the resistance rate of N. gonorrhoeae isolates from Nanjing, Xuzhou and Wuxi against ciprofloxacin has reached 100 % (Table 1
). It is indeed alarming to note such an increase in drug resistance of over 30-fold within 10 years, and this antibiotic should not be recommended for treatment of gonococcal infections in China, especially in the Jiangsu Province. Other antibiotics, such as third- or fourth-generation cephalosporins or spectinomycin, should be considered.
It has been reported that earlier fluoroquinolone-resistant N. gonorrhoeae have mutations in the quinolone-resistant determining region (QRDR) (Belland et al., 1994). While susceptible bacteria have no mutation found in either gyrA or parC (Shultz et al., 2001; Tanaka et al., 2000), when a single mutation in gyrA is found in an isolate, there is an elevation of the fluoroquinolone MIC. In earlier studies by Belland et al. (1994) and Shultz et al. (2001), it has been demonstrated that resistance levels are correlated with sequential changes in gyrA and parC mutations. Higher MICs were obtained when isolates had double gyrA mutations or co-existing parC mutations. Our present study revealed that nearly all the N. gonorrhoeae isolates were fluoroquinolone resistant (Table 1), and among the 54 isolates analysed for mutations, all demonstrated a change of Ser-91Phe in their gyrA gene, and all except one demonstrated a change in position 95 of the amino acid sequence (Table 2).
It was also demonstrated that most of the isolates with gyrA mutations that had co-existing parC mutations were more resistant than those without parC mutations (P<0.05; Table 3). Mutations in the parC gene were found to be mainly caused by amino acid changes in position 86 to 91, with 18 types (Table 2), excluding silent mutations. Previous studies of GyrA and ParC in N. gonorrhoeae have reported 12 amino acid changes in GyrA, including Ala-67Ser, Ala-75Ser, Ala-84Pro, His-88Gln, Ser-91Phe, Ser-91Tyr, Ser-91Cys, Asp-95Gly, Asp-95Ala, Asp-95Asn, Asn-103Ile and Val-120Leu; and 16 amino acid changes in ParC, including Gly-85Cys, Asp-86Asn, Ser-87Ile, Ser-87Asn, Ser-87Arg, Ser-88Arg, Ser-88Pro, Ser-88Thr, Glu-91Lys, Glu-91Gly, Glu-91Gln, Ala-92Gly, Phe-100Leu, Phe-100Tyr, Arg-116His and Arg-116Leu (Chaudhry et al., 2002; Shultz et al., 2001; Su & Lind, 2001; Tanaka et al., 1998, 2000; Trees et al., 1999; Yoo et al., 2004). Our results concurred with most of the findings reported in the literature; in addition, we have found two new mutation sites of Ala-92Pro and Gln-102His in gyrA, and a new mutation of Ala-29Pro in parC (Table 2). We would speculate that there might be geographical differences among resistant isolates of N. gonorrhoeae (Shultz et al., 2001; Yong et al., 2004).
We further analysed the correlation between the number of mutation sites, their position, and the MIC results of the two fluoroquinolones. Through multi-regression analysis, it was revealed that the positions of mutations in the gyrA and parC genes significantly influenced resistance levels against fluoroquinolones, especially the amino acid changes Asp-95Ala in GyrA, and Ser-87Arg and Ser-87Asn in ParC (P<0.05). Since the amino acid change Ser-91Phe in GyrA was found in all isolates, its effect on fluoroquinolones was not measurable through multi-regression analysis due to the lack of a control group. However, correlation of such a mutation to the MIC for fluoroquinolones has previously been demonstrated to be significant (Chaudhry et al., 2002; Shultz et al., 2001; Su & Lind, 2001; Tanaka et al., 1998, 2000; Trees et al., 1999; Yoo et al., 2004). Although the detection of mutations in gyrB and parE was not performed in our study, an increase in the number of mutations would result in higher MICs for ciprofloxacin.
Our findings also revealed variations between mutations and levels of fluoroquinolone resistance in N. gonorrhoeae. Isolates with the same mutations demonstrated different MICs, and isolates with different mutations could give identical MIC results. It is highly likely that fluoroquinolone resistance involves mechanism(s) other than mutations in the gyrA and/or parC genes, as found with other bacterial species, which would require further investigation.
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INTRODUCTION
METHODS
