JAC Advance Access originally published online on February 28, 2007
Journal of Antimicrobial Chemotherapy 2007 59(4):803-804; doi:10.1093/jac/dkm016
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Correspondence |
Mutations in gyrA and parC in ciprofloxacin-resistant Neisseria gonorrhoeae in Kuala Lumpur, Malaysia
Department of Medical Microbiology, Faculty of Medicine, University Malaya, 50603 Kuala Lumpur, Malaysia
* Corresponding author. Tel: +60-3-79676660; Fax: +60-3-79584844; E-mail: wicsam{at}doctors.net.uk
Keywords: N. gonorrhoeae , DNA topoisomerases , resistance , fluoroquinolones
We read with interest the recent report on quinolone-resistant Neisseria gonorrhoeae in Shanghai.1 Ciprofloxacin resistance in N. gonorrhoeae is a serious problem worldwide, especially in Southeast Asia.2 In Malaysia, resistance rates to ciprofloxacin, penicillin and tetracycline have previously been reported to be > 50%.2 At our teaching hospital in Kuala Lumpur, Malaysia, ciprofloxacin resistance rates were 41.4% in 2006. Resistance is mainly caused by mutations in the quinolone resistance determining regions (QRDRs) of the gyrA gene, encoding DNA gyrase subunit A, and parC, encoding topoisomerase IV. The types of gyrA and parC mutations vary geographically, with novel or unusual substitutions reported in some countries.3–6 This study aimed to characterize, for the first time, gyrA and parC mutations in ciprofloxacin-resistant N. gonorrhoeae from Malaysia.
In total, we analysed 18 ciprofloxacin-resistant and 4 ciprofloxacin-susceptible clinical strains isolated in 2004 and 2005. MICs were determined by Etests (AB Biodisk, Solna, Sweden). Susceptible strains had an MIC 
0.06 mg/L, whereas resistant strains had an MIC 
1 mg/L. N. gonorrhoeae ATCC 49226 was used as a control. The QRDRs of gyrA and parC genes of the isolates were amplified using previously described primers, with products corresponding to amino acids 54–146 of the GyrA protein and amino acids 56–140 of the ParC protein.3 PCR reactions were carried out in a 50 µL mixture containing 25 µL of PCR Master Mix (Promega, Madison, WI, USA), 12.5 pmol of each primer and 2 µL of DNA, extracted by boiling. The reaction conditions were 94°C for 1 min, then 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 45 s. The GeneAll PCR SV kit (General Biosystem, Seoul, Korea) was used for DNA purification. Sequencing was performed on an ABI 3730XL autosequencer (Applied Biosystems, Foster City, CA, USA) by Macrogen, Inc. (Seoul, Korea). Sequences were compared with a wild-type strain of N. gonorrhoeae (GenBank accession numbers U08817
[GenBank]
and U08907
[GenBank]
).
The MICs and QRDR mutations for the 22 isolates are shown in Table 1. The MICs of the resistant isolates ranged from 1 to 4 mg/L with a median of 2 mg/L. Nineteen out of 22 strains, including all the susceptible strains, had a silent mutation in parC at codon 131 (CTC to CTG). Only one susceptible strain had a non-silent mutation, which was a single substitution in gyrA. Another silent parC mutation, at codon 104 (TAT to TAC), was seen in a single resistant strain. Both silent mutations have previously been described, although their significance is unknown.7 All the resistant strains had at least two gyrA mutations and at least one mutation in parC, except for one strain with no parC mutations. This is consistent with previous findings that gyrA mutations are necessary for quinolone resistance, whereas parC mutations appear to be complementary, as resistant strains do not carry parC mutations alone.3–6 Some studies further suggest that additional parC mutations facilitate increasing levels of resistance,5 but our study did not support this. For example, one isolate with an MIC of 4 mg/L had two gyrA mutations only, whereas another isolate with an MIC of 1 mg/L had two gyrA mutations and one parC mutation. Other studies have also found no correlation between increasing QRDR mutations and MICs, suggesting that, in some populations, other mechanisms contribute towards quinolone resistance.6
|
One isolate had a novel mutation in gyrA (Ala-92
Ser), along with mutations at positions 91 and 95. Isolates with triple gyrA mutations have been rarely described.1 Previous reports of mutation at position 92 in gyrA have resulted in a different substituted amino acid (Ala-92 Pro).1 With the increasing use of non-culture molecular methods for the diagnosis of gonorrhoea, detailed knowledge of the range of mutations seen globally is essential to accurately detect antimicrobial resistance.
The commonest combination of mutations, seen in 11 (61%) strains, involved two mutations in gyrA (Ser-91 Phe and Asp-95 Gly) and one mutation in parC (Asp-86 Asn); of these, 9 had an additional silent mutation at codon 131 of parC. Reported patterns of QRDR mutations vary between countries. This pattern of mutation positions, involving gyrA positions 91 and 95 and parC position 86, was also predominant in Austria, Philippines, Thailand, Denmark and Hawaii, but was not seen in India and Japan.4 The geographic variations may be due to clonal or polyclonal spread of different local or imported strains.5,6 Typing methods such as PFGE may help elucidate the molecular epidemiology and transmission networks of quinolone-resistant N. gonorrhoeae.
In conclusion, quinolone-resistant N. gonorrhoeae at our hospital have mutation patterns similar to some countries yet distinct from others and demonstrate no correlation between MICs and number of mutations. We also describe a novel gyrA mutation. Although limited by the small number of isolates, this study enhances the existing knowledge of quinolone-resistant N. gonorrhoeae.
None to declare.
Acknowledgements
Preliminary results from this study were presented at the Fourteenth International Union Against Sexually Transmitted Infections-Asia Pacific Conference on STIs and HIV/AIDS, Kuala Lumpur, Malaysia, 2006. We thank Y. F. Ngeow for providing the Etests and N. K. Palanisamy, E. H. Wong and N. S. Sabet for technical assistance. This study was funded in part by a PJP grant F0712/2005B from the University Malaya, Malaysia.
References
1
Yang Y, Liao M, Gu WM, et al. (2006) Antimicrobial susceptibility and molecular determinants of quinolone resistance in Neisseria gonorrhoeae isolates from Shanghai. J Antimicrob Chemother 58:868–72.
2 The WHO Western Pacific Gonococcal Antimicrobial Surveillance Programme. (2003) Surveillance of antibiotic resistance in Neisseria gonorrhoeae in the WHO Western Pacific Region, 2002. Commun Dis Intell 27:488–91.[Medline]
3
Tanaka M, Nakayama H, Haraoka M, et al. (2000) Antimicrobial resistance of Neisseria gonorrhoeae and high prevalence of ciprofloxacin-resistant isolates in Japan, 1993 to 1998. J Clin Microbiol 38:521–5.
4 Uthman A, Heller-Vitouch C, Stary A, et al. (2004) High frequency of quinolone-resistant Neisseria gonorrhoeae in Austria with a common pattern of triple mutations in GyrA and ParC genes. Sex Transm Dis 31:616–8.[Web of Science][Medline]
5 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]
6 Trees DL, Sirivongrangson P, Schultz AJ, et al. (2002) Multiclonal increase in ciprofloxacin-resistant Neisseria gonorrhoeae. Sex Transm Dis 29:668–93.[CrossRef][Web of Science][Medline]
7
Chaudhry U, Ray K, Bala M, et al. (2002) Mutation patterns in gyrA and parC genes of ciprofloxacin resistant isolates of Neisseria gonorrhoeae from India. Sex Transm Infect 78:440–4.
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