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JAC Advance Access originally published online on July 30, 2006
Journal of Antimicrobial Chemotherapy 2006 58(4):868-872; doi:10.1093/jac/dkl301
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Antimicrobial susceptibility and molecular determinants of quinolone resistance in Neisseria gonorrhoeae isolates from Shanghai

Yang Yang1, Mingmin Liao2, Wei-Ming Gu1, Kelli Bell2, Lei Wu1, Nelson F. Eng2, Chu-Guang Zhang1, Yue Chen3, Ann M. Jolly3 and Jo-Anne R. Dillon2,4,*

1 Shanghai Skin Disease and STD Hospital, Shanghai China 2 Vaccine and Infectious Disease Organization, University of Saskatchewan Saskatoon, Canada 3 Department of Epidemiology and Community Medicine, Faculty of Medicine, University of Ottawa Ottawa, Canada 4 Department of Biology, University of Saskatchewan Saskatoon, Canada

*Corresponding author. Tel: +1-306-966-4232; Fax: +1-306-966-8839; E-mail: j.dillon{at}usask.ca

Received 9 January 2006; returned 28 February 2006; revised 29 June 2006; accepted 5 July 2006


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Objectives: To determine the antimicrobial susceptibility of Neisseria gonorrhoeae from Shanghai and to type the quinolone resistance-determining regions (QRDRs) of ciprofloxacin-resistant isolates.

Methods: N. gonorrhoeae isolates (n = 159) were consecutively collected from male patients in Shanghai and examined for their antimicrobial susceptibilities to penicillin, tetracycline, ciprofloxacin, spectinomycin and ceftriaxone. The mutation profiles of the QRDRs of gyrA and parC were determined for 103 isolates including one susceptible isolate and one isolate with intermediate levels of susceptibility to ciprofloxacin.

Results: High percentages of the 159 isolates were resistant to ciprofloxacin (98.7%), penicillin (93.1%) and tetracycline (56.5%). Penicillinase-producing N. gonorrhoeae (PPNG, 37.8%) or penicillinase-producing/tetracycline-resistant N. gonorrhoeae (PP/TRNG, 13.8%) accounted for 51.6% of the isolates. Chromosomal resistance to penicillin was observed in 41.5% of the isolates. Tetracycline resistance was noted in 56.5% of the isolates with 20.1% carrying plasmid-mediated resistance and 36.4% being chromosomally resistant. All isolates were susceptible to ceftriaxone and spectinomycin, although a trend to decreased susceptibility was noted. QRDR mutations were observed in the 101 ciprofloxacin-resistant isolates and the one ciprofloxacin-intermediate isolate, in contrast to the ciprofloxacin-susceptible isolate tested. Mutations in the QRDRs comprised four predominant (65.0% of the 103 isolates) patterns of a total of 19 patterns. Mutations in parC were significantly associated with higher MICs of ciprofloxacin.

Conclusions: Spectinomycin and ceftriaxone are currently recommended for the treatment of gonorrhoea in Shanghai. Although the present study indicates that these antimicrobials should remain effective, the identification of isolates with decreased susceptibility underscores the importance of ongoing antimicrobial susceptibility surveillance to monitor and respond to the emergence of resistant isolates.

Keywords: N. gonorrhoeae , gonorrhoea , resistance mechanisms


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The inexorable development of resistance by Neisseria gonorrhoeae isolates to antibiotics has constrained the number of antibiotics that are clinically effective and relevant. The ineffective or inadequate treatment of gonococcal infections inevitably contributes to gonococcal disease complications disproportionately affecting women and their reproductive health, as well as to the further transmission of gonococcal infections.1 Gonococcal resistance to antimicrobial agents is produced by several mechanisms. Plasmid-mediated resistance may confer resistance to penicillin (i.e. penicillinase-producing N. gonorrhoeae, PPNG) or to tetracycline (i.e. tetracycline-resistant N. gonorrhoeae, TRNG), while chromosomally mediated (CM) resistance is caused by chromosomal mutations at various loci. The accumulation of chromosomal mutations may confer resistance to multiple antibiotics or confer higher levels of resistance to single antibiotics.1

Quinolone-resistant N. gonorrhoeae (QRNG) isolates are now prevalent in many countries including China.2–4 This resistance is primarily caused by point mutations in gyrA and parC at loci described as quinolone resistance-determining regions (QRDRs).5,6 Specific mutations in QRDRs in N. gonorrhoeae isolated from Shanghai have not previously been determined. In the present study, we investigated the antimicrobial susceptibility and the molecular mechanisms of quinolone resistance of N. gonorrhoeae isolates from Shanghai.


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Isolation, identification and growth of N. gonorrhoeae isolates

Clinical N. gonorrhoeae isolates (n = 159) were consecutively collected from male patients at the Shanghai Skin Disease and STD Hospital between November 2004 and May 2005. Urogenital specimens were collected using sterile Dacron swabs, which were streaked onto Thayer–Martin (T–M) medium (Oxoid; distributed by GuangZhou LOSO Science Ltd, Shanghai, China) supplemented with 1% IsoVitalex (Oxoid; GuangZhou LOSO Science Ltd). Inoculated T–M plates were incubated at 35°C in a humid, 5% CO2 environment, for 24–48 h. N. gonorrhoeae was identified using criteria including the oxidase test, Gram stain and glucose utilization tests.2 Isolates were subcultured on GC agar base (Oxoid; GuangZhou LOSO Science Ltd) supplemented with 1% IsoVitalex (GCBI) prior to antimicrobial susceptibility testing. Isolates were stored at –80°C in brain heart infusion medium (Difco; distributed by Shanghai Chemical Reagent Co., China National Medicine Group, Shanghai, China) containing 20% glycerol. porB DNA sequence analysis of the 159 isolates was performed and indicated that the isolates were not derived from a single clone (data not shown).

Antimicrobial susceptibility testing

The MICs for N. gonorrhoeae of penicillin (0.008–64.0 mg/L), tetracycline (0.008–64.0 mg/L), ciprofloxacin (0.002–64.0 mg/L), spectinomycin (2.0–256.0 mg/L) and ceftriaxone (0.002–2.0 mg/L) were determined by the agar dilution method.7 Antimicrobial agents were purchased from the Shanghai ANPEL Scientific Instrument Co. Ltd (Shanghai, China; distributors of Sigma-Aldrich, USA). Each MIC determination was performed in duplicate and included WHO reference strains A, B, C, D and E for each test.8 Gonococcal inocula were prepared by suspending an overnight GCBI culture in 0.9% saline to a turbidity equivalent to that of a 0.5 McFarland standard (Remel, Lenexa KS, USA). The inoculum was adjusted to a final concentration of 107 cfu/mL, and ~2 µL was delivered onto antibiotic-containing and control GCBI plates using a multispot inoculator (A400 Denley, UK). Inoculated plates were incubated, as described above, for 18–24 h. ß-Lactamase production was determined for all isolates using the chromogenic cephalosporin test (Oxoid; GuangZhou LOSO Science Ltd). Interpretative MIC criteria7 included the following resistance classifications (isolates may carry combinations of resistance phenotypes, e.g. PPNG-CMTR-CipR, where CipR stands for ciprofloxacin resistant): PPNG (ß-lactamase positive), TRNG (isolates having tetracycline MICs ≥16 mg/L), PP/TRNG (ß-lactamase positive and MICs of tetracycline ≥16 mg/L), CMPR (non-PPNG isolates with penicillin MICs ≥2 mg/L), CMTR (non-TRNG isolates with tetracycline MICs ≥2–8 mg/L), CMRNG (non-PPNG and non-TRNG isolates with penicillin MICs ≥2 mg/L and tetracycline MICs ≥2–<16.0 mg/L).

QRDR sequence analysis

The DNA sequences of the QRDRs of 103 isolates, comprising 101 ciprofloxacin-resistant isolates, 1 intermediate isolate and 1 susceptible isolate, were determined. These isolates were selected based on their resistance phenotypes and there was no significant difference (P > 0.05) in the resistance classification percentage (i.e. %PPNG, %CMTR, etc.) between these isolates and the 159 original isolates tested. Gonococcal DNA was extracted using the Genomic DNA Purification Kit (Shanghai Promega Biological Products Ltd, Shanghai, China). The QRDRs of gyrA and parC were amplified using PCR (Perkin Elmer 9600 Thermo Cycler; Perkin Elmer, Wellesley, MA USA) as described previously.5,6 Primers5,6 were purchased from Invitrogen Canada (Burlington, Ontario, Canada). Following the purification of PCR products (PCR Purification Kit; Qiagen, Mississauga, Ontario, Canada) their DNA sequences were determined (Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, Canada) using the same primers.5,6 DNA sequences were translated using Proteomics and Sequence Tools (http://ca.expasy.org/, last accessed on 25 June 2006). QRDRs from N. gonorrhoeae FA1090 were used as prototypes (GenBank #P48371 for gyrA and #U08907 for parC). Correlation coefficients between QRDR mutations and ciprofloxacin MICs were determined using the F-test (Microsoft Excel).


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The antimicrobial susceptibilities of 159 N. gonorrhoeae isolates are summarized in Table 1. Penicillin MICs ranged from 0.5 to ≥64 mg/L, with an MIC50 of 32 mg/L and an MIC90 of ≥64 mg/L; 93.1% of the isolates were resistant to penicillin and 6.9% demonstrated intermediate levels of susceptibility (MICs 0.5–1.0 mg/L). Plasmid-mediated penicillin resistance was observed in 51.6% of the isolates with 37.8% (n = 60) being PPNG and 13.8% (n = 22) being PP/TRNG. Chromosomal penicillin resistance was observed in 41.5% of the isolates with 23.3% (n = 37) classified as CMPR and 18.2% (n = 29) as CMRNG. The total burden of tetracycline resistance was 56.5% and included isolates classified as TRNG (6.3%), PP/TRNG (13.8%), CMTR (18.2%) and CMRNG (18.2%). Isolates had tetracycline MICs extending from 0.06 to ≥64 mg/L with an MIC50 of 2 mg/L and an MIC90 of 32 mg/L. The ciprofloxacin MIC range of the isolates was 0.06–≥64 mg/L (MIC50 and MIC90 values of 8 and 32 mg/L, respectively); 98.7% of the isolates were ciprofloxacin resistant while 1 (0.6%) isolate was susceptible, and 1 (0.6%) was classified at an intermediate (MIC 0.5 mg/L) susceptibility level. All isolates were susceptible to spectinomycin (MIC < 256 mg/L) with 98.1% having MICs classified as susceptible and 1.9% (n = 3) demonstrating intermediate (MIC = 64 mg/L) levels of susceptibility. The isolates were also susceptible to ceftriaxone (MIC range: 0.004–0.25 mg/L; MIC50 and MIC90 values of 0.03 and 0.125 mg/L, respectively). In all, nine categories of resistance phenotypes were identified with the majority comprising PPNG-CipR (20.8%), CMPR-CipR (16.4%), CMRNG-CipR (18.2%), PPNG-CMTR-CipR (17.6%) and PP/TRNG-CipR (13.2%).


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  		Table 1. MICs of five antimicrobial agents for 159 N. gonorrhoeae isolates from Shanghai

 
The QRDR mutation profiles of 103 isolates are shown in Table 2. The single ciprofloxacin-susceptible isolate tested did not carry mutations in its QRDR of gyrA or parC (mutation pattern P0), while the ciprofloxacin-intermediate isolate carried an Asp-95->Ala mutation in gyrA and a Ser87->Asn mutation in parC (pattern P1). All ciprofloxacin-resistant isolates carried a Ser-91->Phe mutation in the QRDR of gyrA, and some isolates also contained Ala-92->Pro (3/103, 2.9%) and Asp-95->Ala/Gly (85/103, 82.5%) mutations. Single, double or triple mutations in gyrA accounted for 15.5%, 82.5% and 1.0% of mutations in gyrA, respectively. parC mutations occurred in 74.8% of the isolates tested at residues Ser-87 and Glu-91 including Ser-87->Arg (45/103, 43.7%) or Ser-87->Asn/Ile/Cys (27/103, 26.2%). Single substitutions at Ser-87 were more commonly found in isolates with parC mutations (69/103, 67.0%), while mutations at Glu-91 accounted for 7.8% of the isolates (8/103). Mutations in gyrA were not correlated with ciprofloxacin MIC values, whereas a significant correlation (P < 0.001) between parC mutation rates and MIC values of ciprofloxacin was observed (data not shown). Mutations in QRDRs were grouped into 19 patterns (Table 2). The most predominant patterns, accounting for 65.0% of the 103 isolates tested, included P3, P8, P9 and P15, with mutations at Ser-91 and Asp-95 of gyrA and a single or no mutation at Ser-87 of parC. Isolates with patterns P2, P3, P4 and P16 (24.3%) contained gyrA but no parC mutations. Other patterns were characterized by one substitution in both gyrA and parC (P1, P5 and P6), two mutations in gyrA and one mutation in parC (P7, P8, P9, P10, P11, P14, P15 and P17), double mutations in both gyrA and parC (P12 and P13), three gyrA mutations and one parC mutation (P18) or one gyrA mutation and two parC mutations (P19).


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  		Table 2. gyrA and parC QRDR mutation patterns in 103 N. gonorrhoeae isolates

 

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The burden of antimicrobial resistance of N. gonorrhoeae isolates has increased in China since the mid-1990s;8 between 1993 and 1998, 66.7% of 3186 isolates tested were resistant to penicillin and 92.0% were resistant to tetracycline. Ciprofloxacin resistance increased from 15.5% of isolates tested in 1993 to 55.8% in 1998. By 2004 ciprofloxacin-resistant isolates comprised 94.3% (n = 1203) of isolates tested.9 In comparison, in the present study, the percentage of ciprofloxacin-resistant isolates included over 98% of the isolates, the total burden of penicillin resistance was over 90%, while the burden of tetracycline resistance was 56.5% of the isolates. The percentage of either PPNG or TRNG is consistent with other reports from China in which PPNG comprised 48.8% of 1002 isolates and TRNG included 34.2% of 1202 isolates9 as compared with 51.6% of isolates typed as being PPNG (and PP/TRNG) and 20.1% characterized as TRNG (and PP/TRNG) in the present study.

The unremitting development of resistance to multiple antibiotics in N. gonorrhoeae isolates has provoked the ongoing modification of treatment guidelines in China; the recommended treatment of choice for uncomplicated gonococcal infections changed from penicillin, in 1989, to ciprofloxacin in 1995, and subsequently to ceftriaxone or spectinomycin in 2000 (Diagnostic criteria and management of gonorrhoea, Ministry of Health, People's Republic of China, 2000). Although none of the circulating isolates tested in the present study was resistant to either spectinomycin or ceftriaxone, a small percentage was characterized as having intermediate levels of susceptibility to spectinomycin. Importantly, the MIC90 of ceftriaxone was one dilution lower than the concentration defining the limits of susceptibility7 and over 10% of the isolates had MICs which were within two dilutions of this critical concentration. Other reports from China have also documented isolates with decreased susceptibility to ceftriaxone.8,9

The majority of the QRDR mutation patterns included double mutations of gyrA (Ser-91->Phe and Asp-95->Ala/Gly) combined with a single (Ser-87->Arg/Asn) or no mutation of parC (Table 2). Although these and other mutation patterns have also been observed by other investigators from different countries,10,11 the predominant mutational patterns varied from those reported in other regions. The presence of parC mutations was significantly associated with higher ciprofloxacin MIC values, suggesting that parC mutations proportionately reduce susceptibility and enhance resistance to quinolones in N. gonorrhoeae isolates. The absence of parC mutations in some ciprofloxacin-resistant isolates indicates that it is not always the primary determinant specifying gonococcal resistance to ciprofloxacin. gyrA mutations were present in all ciprofloxacin-resistant isolates but not in the one ciprofloxacin-susceptible isolate, indicating that gyrA mutations are necessary for the quinolone resistance phenotype. Thus, the present study quantitatively supports the hypothesis that gyrA mutations determine whether a gonococcal isolate is resistant to quinolones, while a mutation in parC is correlated with high-level quinolone resistance.11

The study combines antimicrobial susceptibility determinations with molecular-based analysis of ciprofloxacin resistance in N. gonorrhoeae isolated in Shanghai. While ceftriaxone and spectinomycin should be effective antibiotics for the treatment of gonorrhoea in Shanghai, continued monitoring of gonococcal susceptibility to these agents is crucial given the decreased susceptibilities of some isolates. The identification of ciprofloxacin-resistant isolates coupled with the characterization of QRDR mutation patterns will serve as a baseline to better appreciate gonococcal transmission patterns and the evolution of resistant clones in Shanghai.


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None to declare.


   Acknowledgements
 
Ethical approval for this study was obtained from the Ottawa Hospital Research Ethics Board and the Ethics Committee of Shanghai Municipal Bureau of Public Health. This work was supported by the Canadian Institutes of Health Research (CIHR) (Grant #OPC-67017). Dr Yang was a Visiting Scholar at the University of Saskatchewan with J. R. D. and N. F. E. was funded by a CIHR doctoral scholarship.


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1 Dillon JR and Pagotto F. (1999) Importance of drug resistance in gonococci: from mechanisms to monitoring. Curr Opin Infect Dis 12:35–40.[Medline]

2 The WHO Western Pacific Gonococccal Antimicrobial Surveillance Program. (2005) Surveillance of antibiotic resistance in Neisseria gonorrhoeae in the WHO Western Pacific Region, 2003. Commun Dis Intell 29:62–4.[Medline]

3 Knapp JS, Fox KK, Trees DL, et al. (1997) Fluoroquinolone resistance in Neisseria gonorrhoeae. Emerg Infect Dis 3:33–39.[Medline]

4 Tompkins JR and Zenilman JM. (2001) Quinolone resistance in Neisseria gonorrhoeae. Curr Infect Dis Rep 3:156–161.[Medline]

5 Trees DL, Sandul AL, Whittington WL, et al. (1998) Identification of novel mutation patterns in the parC gene of ciprofloxacin-resistant isolates of Neisseria gonorrhoeae. Antimicrob Agents Chemother 42:2103–2105.[Abstract/Free Full Text]

6 Belland RJ, Morrison SG, Ison C, et al. (1994) Neisseria gonorrhoeae acquires mutations in analogous regions of gyrA and parC in fluoroquinolone-resistant isolates. Mol Microbiol 14:371–80.[Web of Science][Medline]

7 National Committee for Clinical Laboratory Standards. (2002) Performance Standards for Antimicrobial Susceptibility Testing: Twelfth Informational Supplement M100-S12 (NCCLS, Villanova, PA, USA).

8 Ye S, Su X, Wang Q, et al. (2004) Fifteen year monitoring of drug resistance of gonococci. Chinese J Human Sexuality 13:1–3.

9 The WHO Western Pacific Surveillance Program. (2006) Surveillance of antibiotic resistance in Neisseria gonorrhoeae in the WHO Western Pacific Region, 2004. Commun Dis Intell 30:129–32.[Medline]

10 Giles JA, Falconio J, Yuenger JD, et al. (2004) Quinolone resistance-determining region mutations and por type of Neisseria gonorrhoeae isolates: resistance surveillance and typing by molecular methodologies. J Infect Dis 189:2085–93.[CrossRef][Web of Science][Medline]

11 Trees DL, Sandul AL, Peto-Mesola V, et al. (1999) Alterations within the quinolone resistance-determining regions of gyrA and parC of Neisseria gonorrhoeae isolated in the Far East and the United States. Int J Antimicrob Agents 12:325–32.[CrossRef][Web of Science][Medline]


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