Fluoroquinolone-resistant Neisseria gonorrhoeae isolates from Russia: molecular mechanisms implicated

V. A. Vereshchagin1,*, E. N. Ilina1, M. V. Malakhova1, M. M. Zubkov2, S. V. Sidorenko3, A. A. Kubanova2 and V. M. Govorun1

1 Institute of Physico-Chemical Medicine, Malaya Pirogovskaya st., 1a, 119992, Moscow; 2 Central Research Institute of Dermatology and Venereology, Moscow; 3 National Research Centre for Antibiotics, Moscow, Russia

Received 27 October 2003; returned 4 December 2003; revised 14 January 2004; accepted 15 January 2004


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: During a longitudinal study of the prevalence of antimicrobial resistance in Neisseria gonorrhoeae, a number of high-level fluoroquinolone-resistant isolates were obtained from the sexually transmitted diseases clinic in the Moscow region in 2002. The aim of the present study was to determine the molecular mechanisms of resistance and to assess the clonal relationship of these strains

Methods: For the 32 clinical strains of N. gonorrhoeae studied, the MIC values were determined for four fluoroquinolones. The gyrA, parC, por and mtrR genes were studied for the presence of mutations associated with fluoroquinolone resistance.

Results: We detected strains of N. gonorrhoeae showing high-level resistance to fluoroquinolones (21 strains, with MICs 1–32 mg/L). Mutations in gyrA and parC known to cause fluoroquinolone resistance were detected in a majority of strains. There were four strains (among 21) without known changes in gyrA and parC. However, amino acid changes in the Por protein and mutations in the promoter or encoding region of the mtrR gene were detected in three of them. One strain had no alteration in gyrA, parC, por or mtrR.

Conclusions: The present study documents the first case of fluoroquinolone-resistant N. gonorrhoeae in Russia.

Keywords: N. gonorrhoeae, fluoroquinolones, mechanisms of resistance


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Quinolones became available for treatment of gonorrhoea in the mid-1980s and since 1989 they have been used as a single-dose oral therapy. Quinolone resistance in Neisseria gonorrhoeae has been reported mostly in the Far East, the Philippines, Hong Kong and Japan. Less quinolone resistance has been reported in Australia, the USA and Europe.

Mutations in the gyrA and parC genes, which lead to alterations in the GyrA and ParC subunits of DNA gyrase and topoisomerase, respectively, have a central role in resistance to fluoroquinolones.1,2

Resistance to various antibacterial agents: penicillins, tetracyclines, macrolides, fluoroquinolones and hydrophobic antibacterial agents produced by the host itself (fatty acids, antibacterial peptides) can also be predetermined by mutations in the promoter or encoding region of the mtrR gene, which leads to increased action by the MtrC-MtrD-MtrE efflux system.3,4

Strains of N. gonorrhoeae resistant to ciprofloxacin5 and norfloxacin6 were shown to have decreased bacterial cell permeability to fluoroquinolones. This is possibly connected with a series of amino acid changes in the Por protein, as is the case with resistance to penicillin and tetracycline.7

According to previous studies of N. gonorrhoeae in Russia, only sporadic isolates with low-level ciprofloxacin resistance (MICs 0.125–0.5 mg/L) were found in 1998.8 However, larger numbers of isolates with high-level fluoroquinolone resistance were found in the sexually transmitted diseases clinic in the Moscow region during a surveillance programme established in Russia in 2002.

The aim of the present work was to determine the molecular mechanisms of resistance and to assess the clonal relationship of strains.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strains

Thirty-two clinical strains of N. gonorrhoeae isolated from patients with uncomplicated gonorrhoea in Moscow and the Moscow region in 2002 were studied.

Antimicrobial susceptibility testing

MICs of ciprofloxacin and moxifloxacin (both from Bayer AG, Germany), and levofloxacin and ofloxacin (both from Aventis Pharma S.A., France) were determined by an agar dilution technique9 using GC agar (bioMérieux, France) supplemented with PolyViteX (bioMérieux, France). N. gonorrhoeae ATCC 49226 was used as a control. All tests were performed in triplicate. The antimicrobial susceptibility was judged according to breakpoint criteria defined by the NCCLS.9 The respective concentrations for susceptible/intermediate/resistant breakpoints were <=0.06/0.125–0.5/>=1 mg/L for ciprofloxacin and <=0.25/0.5–1/>=2 mg/L for ofloxacin. NCCLS breakpoints for moxifloxacin and levofloxacin have not been determined.

DNA isolation and amplification of N. gonorrhoeae genome fragments

Total DNA of N. gonorrhoeae was isolated according to the method of Boom et al.10 Previously recommended primers1,2,4,11 were used to amplify the quinolone resistance determining regions of the gyrA and parC genes, the promoter and a fragment of the encoding region of the mtrR gene and the por gene. The PCR amplification was carried out in a reaction mixture containing 10 mM Tris-HCl pH 9.0, 50 mM KCl, 2 mM MgCl2, 250 µM each dNTP, 1 unit Taq polymerase (Promega, USA) and 10 pmol of each primer, using the thermocycler ‘Tercyc’ (DNA-Technology, Russia). The products of amplification were analysed in a 2% agarose gel.

Determination of the nucleotide and amino acid sequences

The amplification products were purified on the Wizard PCR Preps DNA Purification System (Promega, USA), for determination of the nucleotide sequences. Sequencing of the gene (por, gyrA, parC, mtrR) fragments was carried out using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit and the ABI Prism 3100 Genetic Analyser (Applied Biosystems, USA; Hitachi, Japan). The full-scale sequence of the por gene was obtained by means of assembling the nucleotide fragments using the software product ‘Vector NTI Suite v. 6.0’ (InforMax, Inc., USA).

The amino acid sequences of GyrA, ParC and MtrR were determined from the known nucleotide sequences using the software ‘Vector NTI Suite v. 6.0’ (InforMax, Inc.).

Por typing of N. gonorrhoeae strains

The Por type of the strains was determined by identifying the obtained nucleotide sequences of the por genes in the EMBL (European Molecular Biology Laboratory) and NCBI (National Centre for Biotechnology Information) databases.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Table 1 shows susceptibility data for 32 N. gonorrhoeae strains with details of Por type, GyrA, ParC, MtrR and Por alterations and mtrR gene promoter mutations.


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Table 1. MICs of fluoroquinolones for 32 strains of Neisseria gonorrhoeae with details of Por type, GyrA, ParC, MtrR and Por changes, and mtrR gene promoter mutations
 
The 32 strains studied can be divided into two groups. Strains in the first group (n = 10) were fully susceptible to fluoroquinolones (MICs were in the range 0.001–0.06 mg/L). Strains in the second group demonstrated different levels of fluoroquinolone resistance (MICs 1–32 mg/L). The majority of them showed high-level resistance (MIC >= 4 mg/L) to all fluoroquinolones studied. One strain demonstrated a ciprofloxacin MIC of 4 mg/L and ‘wild-type’ MICs of other compounds, and a further strain demonstrated low-level levofloxacin resistance (MIC 0.12 mg/L) and susceptibility to the other fluoroquinolones.

Previously, Shultz et al.12 showed that strains having mutations simultaneously in GyrA and ParC exhibited a higher level of resistance than strains having mutations only in GyrA, with the maximal MIC values being shown for the strains having mutations in both GyrA and ParC. According to our study, no such pattern could be discerned (Table 1).

Amino acid substitutions known to lead to development of fluoroquinolone resistance were identified in 17 of 21 phenotypically resistant strains. Among the strains examined, we revealed no single mutations in GyrA and this agrees with the absence of strains with low-level fluoroquinolone resistance; only double GyrA mutations were detected. Double GyrA mutations (Ser-91->Phe, Asp-95->Gly) were combined with the ParC mutation Ser-87->Arg in seven isolates. In addition, double GyrA mutations (Ser-91->Phe, Asp-95->Asn) were combined with the ParC mutation Glu-91->Gly in two strains. In one strain without any GyrA mutations, the ParC mutation Glu-91->Gly was detected. In our opinion, this is the first finding of a clinical isolate with high-level fluoroquinolone resistance due to a single first-step mutation in parC. It was possible to determine five genotypes in the examined isolates, indicating their genetic heterogeneity. Apparently, in the Moscow region, independent processes of selection and accumulation of fluoroquinolone-resistant strains take place.

In cases of fluoroquinolone resistance without mutations in the GyrA and ParC proteins (four strains) one could surmise the existence of unknown alterations in gyrase and topoisomerase genes.

It is proposed that mutations in the promoter and the encoding region of the mtrR gene may contribute to fluoroquinolone resistance in N. gonorrhoeae due to increased expression of the proteins of the MtrCDE system.3 Analysis of the polymorphism of the mtrR gene and its promoter in the examined samples showed that 21 strains had mutations in the promoter and/or substitution at amino acid position 45 of the MtrR protein. The majority of them (20 strains) had only one of the two possible mutations. We showed that eight of 10 fluoroquinolone-susceptible strains also had this mutation (Table 1).

Analysis of the polymorphism of Por at positions 120 and 121 showed that 22 strains had amino acid substitutions in the Por protein that decrease bacterial cell permeability for penicillin and tetracycline.7 No correlation between amino acid substitutions and the level of fluoroquinolone resistance was found.

Thus our data did not confirm the possible role of reduced cell permeability and efflux in N. gonorrhoeae resistance to fluoroquinolones.

Por typing revealed genetic heterogeneity among the isolates studied. Thirty-one strains belonged to the PIB serovar (PIB3, 41.9%; PIB2, 19.4%; PIB5, 12.9%; PIB7, 9.7%; PIB3/5, 6.5%; PIB4, 3.2%; PIB3/6, 3.2%; PIB8, 3.2%) and one strain belonged to the PIA serovar.

This work documents the first detection of quinolone-resistant N. gonorrhoeae in Russia. The results of the genetic analysis lead us to conclude that this fact is not a local phenomenon predetermined by distribution of one or a limited number of clones. Hence, in spite of the limited number of samples, these results are important for planning antibacterial therapy of gonorrhoea in the Moscow region. It should be noted that the number of studied isolates is insufficient to extrapolate results to the whole of Russia. Regional surveillance studies on the prevalence of fluoroquinolone resistance in N. gonorrhoeae are essential.


    Footnotes
 
* Corresponding author. Tel: +7-095-2454236; Fax: +7-095-2464501; E-mail: vereshchagin{at}nm.ru Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Deguchi, T., Yasuda, M., Asano, M. et al. (1995). DNA gyrase mutations in fluoroquinolone-resistant clinical isolates of Neisseria gonorrhoeae. Antimicrobial Agents and Chemotherapy 39, 561–3.[Abstract]

2 . Trees, D. L., Sandul, A. L., Whittington, W. L. et al. (1998). Identification of novel mutation patterns in the parC gene of ciprofloxacin-resistant isolates of Neisseria gonorrhoeae. Antimicrobial Agents and Chemotherapy 42, 2103–5.[Abstract/Free Full Text]

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4 . Lucas, C. E., Balthazar, J. T., Hagman, K. E. et al. (1997). The MtrR repressor binds the DNA sequence between the MtrR and mtrC genes of Neisseria gonorrhoeae. Journal of Bacteriology 179, 4123–8.[Abstract]

5 . Corkill, J. E., Percival, A. & Lind, M. (1991). Reduced uptake of ciprofloxacin in a resistant strain of Neisseria gonorrhoeae and transformation of resistance to other strains. Journal of Antimicrobial Chemotherapy 28, 601–4.[ISI][Medline]

6 . Tanaka, M., Fukuda, H., Hirai, K. et al. (1994). Reduced uptake and accumulation of norfloxacin in resistant strains of Neisseria gonorrhoeae isolated in Japan. Genitourinary Medicine 70, 253–5.[ISI][Medline]

7 . Olesky, M., Hobbs, M. & Nicholas, R. A. (2002). Identification and analysis of amino acid mutations in porin IB that mediate intermediate-level resistance to penicillin and tetracycline in Neisseria gonorrhoeae. Antimicrobial Agents and Chemotherapy 46, 2811–20.[Abstract/Free Full Text]

8 . Sekhine, S. V. & Stratchounski, L. S. (1999). Resistance to antibiotics in Neisseria gonorrhoeae in Russia: there is something to worry about. In Abstracts of the Twenty-first International Congress of Chemotherapy, Birmingham, UK, 1999. Abstract P188. Journal of Antimicrobial Chemotherapy 44, Suppl. A, 81.

9 . National Committee for Clinical Laboratory Standards. (2003). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Sixth Edition: Approved standard M7-A6. NCCLS, Villanova, PA, USA.

10 . Boom, R., Sol, C. J. A., Salimans, M. M. M. et al. (1990). Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology 28, 495–503.[ISI][Medline]

11 . Viscidi, R. P., Demma, J. C., Gu, J. et al. (2000). Comparison of sequencing of the por gene and typing of the opa gene for discrimination of Neisseria gonorrhoeae strains from sexual contacts. Journal of Clinical Microbiology 38, 4430–8.[Abstract/Free Full Text]

12 . Shultz, T. R., Tapsall, J. W. & White, P. A. (2001). Correlation of in vitro susceptibilities to newer quinolones of naturally occurring quinolone-resistant Neisseria gonorrhoeae strains with changes in GyrA and ParC. Antimicrobial Agents and Chemotherapy 45, 734–8.[Abstract/Free Full Text]