Short communicationEmergence of clinical strains of Mycoplasma genitalium harbouring alterations in ParC associated with fluoroquinolone resistance
Introduction
Mycoplasma genitalium was first isolated in urethral cultures from two men with non-gonococcal urethritis (NGU) in 1981 [1]. Mycoplasma genitalium had been proposed as a cause of human NGU, but its role in the aetiology of NGU was not established because of the immense difficulty in isolating it from clinical specimens. Since polymerase chain reaction (PCR)-based assays facilitated detection of M. genitalium in clinical specimens, it has been shown to be significantly associated with NGU in men and with cervicitis, endometritis, salpingitis and pelvic inflammatory diseases in women.
Although clinical data on the treatment of M. genitalium infections are very limited, most therapies for chlamydial infection also appear to be effective against M. genitalium. However, treatment with doxycycline regimens is not so effective [2]. Microbiological eradiation of M. genitalium with a single 1 g dose of azithromycin was reported to be 72–87% [2]. In our previous studies on the treatment of NGU positive for M. genitalium with fluoroquinolones, microbiological eradication of the Mycoplasma with a 7-day regimen of 400 mg/day gatifloxacin was 92% [3]. In other studies, moxifloxacin given at 400 mg daily for 7 days or 10 days was reported to be the only efficient treatment option in cases of treatment failure with doxycycline or azithromycin [4]. Some fluoroquinolones are being proposed as promising agents for the treatment of M. genitalium infections. In Japan, however, multiple low doses of levofloxacin had frequently been used to treat patients with NGU, but microbiological eradication of the Mycoplasma with a 14-day regimen of 300 mg/day levofloxacin was reported to be only 50% [3].
Surveillance for antimicrobial resistance in clinical strains of M. genitalium is crucial for determining subsequent treatment guidelines. However, isolation of M. genitalium in culture remains a labour-intensive and time-consuming task. Therefore, in the present study we attempted to apply a molecular approach that does not involve culturing of M. genitalium and testing of in vitro antimicrobial susceptibility in order to assess fluoroquinolone resistance in clinical strains of M. genitalium.
Fluoroquinolones act by binding to their target enzymes, DNA gyrase and topoisomerase IV, and interfere with DNA replication. DNA gyrase is composed of two GyrA and two GyrB subunits, and topoisomerase IV is composed of two ParC and two ParE subunits. The central mechanism of fluoroquinolone resistance involves alterations of the GyrA subunit of DNA gyrase and/or the ParC subunit of topoisomerase IV in bacterial species, including mycoplasmas and ureaplasmas [5], [6], [7], [8], [9], [10], [11], [12]. Alterations in GyrB and ParE play a complementary role in the development of fluoroquinolone resistance. Therefore, in this study we amplified the quinolone-resistance determining region (QRDR) of the gyrA gene [13] and the analogous region of the parC gene from M. genitalium DNA taken from urine specimens of men with NGU positive for M. genitalium and determined their sequences. Amino acid changes in GyrA and ParC produced by mutations in the QRDRs of the gyrA and parC genes were compared with those previously reported in fluoroquinolone-resistant mutants of other Mycoplasma and Ureaplasma spp. [5], [6], [7], [8], [9], [10], [11], [12].
Section snippets
Patients and urine specimens
This retrospective study was approved by the Institutional Review Board of the Graduate School of Medicine, Gifu University (Gifu, Japan). Urine specimens were collected from men with NGU who visited a urologic clinic (iClinic, Sendai, Japan) between 2006 and 2008 for evaluation of microbial aetiologies of urethritis. Specimens were stored at −70 °C with the informed consent of each patient. All specimens were examined for the presence of Chlamydia trachomatis, M. genitalium, Mycoplasma hominis,
Results
In one specimen, M. genitalium had a T → A transition at nucleotide position 321 in the gyrA gene, resulting in an amino acid change (Phe-108 → Iso) in GyrA, but no mutations in the QRDR of the parC gene (Table 1). In another specimen, the same mutation was observed in the gyrA gene as well as an A → G transition at nucleotide position 290 (Lys-97 → Arg) in the parC gene. In three respective specimens no mutations in the gyrA gene were observed, but a single mutation in the parC gene of a G → A
Discussion
In this study, amino acid changes were found in GyrA and ParC of M. genitalium present in the pre-treatment urine specimens of men with NGU. Amongst these amino acid changes, Ser-83 → Asn, Asp-87 → Tyr and Asp-87 → Val in ParC, corresponding to changes at amino acid positions 80 and 84 in E. coli ParC, respectively, which have been proposed to be related to resistance in bacterial species, including other mycoplasmas and ureaplasmas [5], [6], [7], [8], [9], [10], [11], [12], could be associated with
References (15)
- et al.
A newly discovered mycoplasma in the human urogenital tract
Lancet
(1981) - et al.
A randomized comparison of azithromycin and doxycycline for the treatment of Mycoplasma genitalium-positive urethritis in men
Clin Infect Dis
(2009) - et al.
In vitro activity of fluoroquinolones against Mycoplasma genitalium and their bacteriological efficacy for treatment of M. genitalium-positive nongonococcal urethritis in men
Clin Infect Dis
(2005) - et al.
Azithromycin and moxifloxacin for microbiological cure of Mycoplasma genitalium infection: an open study
Int J STD AIDS
(2008) - et al.
Mutations in the gyrA, parC, and parE genes associated with fluoroquinolone resistance in clinical isolates of Mycoplasma hominis
Antimicrob Agents Chemother
(1999) - et al.
In vitro development of resistance to six and four fluoroquinolones in Mycoplasma pneumoniae and Mycoplasma hominis, respectively
Antimicrob Agents Chemother
(2005) - et al.
Molecular characterization and typing of enrofloxacin-resistant clinical isolates of Mycoplasma gallisepticum
Avian Dis
(2008)