Analysis of the gyrA and parC genes of Mycoplasma genitalium detected in first-pass urine of men with non-gonococcal urethritis before and after fluoroquinolone treatment

Takashi Deguchia,*, Shin-Ichi Maedab, Masayoshi Tamakib, Takashi Yoshidac, Hiroaki Ishikoc, Masayasu Itoa, Shigeaki Yokoia, Yoshihito Takahashia and Satoshi Ishiharaa

a Department of Urology, Gifu University School of Medicine, Gifu; b Department of Urology, Toyota Memorial Hospital, Toyota; c Infectious Disease Laboratory, Mitsubishi Kagaku Bio-Clinical Laboratories, Inc., Tokyo, Japan

Sir,

Although Mycoplasma genitalium has been proposed as a cause of human non-gonococcal urethritis (NGU), the role of the mycoplasma in the aetiology of NGU has not been established because of the immense difficulty in isolating it from clinical samples. PCR-based assays have facilitated detection of M. genitalium in clinical specimens, and a significant association between M. genitalium and NGU has been demonstrated. Because isolation of M. genitalium in culture is still difficult, the antibiotic susceptibility of current clinical strains of M. genitalium is poorly understood. In this study, we amplified the quinolone resistance-determining region (QRDR) of the gyrA gene and the analogous region of the parC gene of M. genitalium from first-pass urine samples of men with M. genitalium PCR-positive NGU, and examined the genes for the presence of fluoroquinolone resistance-associated mutations.

Study participants were six men with M. genitalium- positive NGU, in whom fluoroquinolone treatment failed to eradicate the mycoplasma from the urethra.1 None of the men had received antibiotic treatment prior to admission to the clinic. They were treated with levofloxacin at a dosage of 100 mg tds for 14 days. First-pass urine was tested for the presence of M. genitalium by PCR amplification of the 16S rRNA gene and phylogenetic analysis. One man had evidence of inflammation and was positive for M. genitalium after levofloxacin treatment. The remaining five men experienced alleviation of symptoms and no evidence of inflammation, but they remained positive for the mycoplasma after treatment.

DNA samples were purified from pre- and post-treatment first-pass urine samples that had been PCR-positive for the mycoplasma. From these DNA samples, portions of the gyrA gene (nucleotides 172–402)2 and the parC gene (nucleotides 164–483)3 of M. genitalium were amplified by PCR. The PCR primers for the gyrA gene were MG-GYRA-A (5'-CGTCGTGTTCTTTATGGTGC-3') and MG-GYRA-B (5'-ATAACGYYGTGCAGCAGGTC-3'); PCR primers for the parC gene were MG-PARC-A (5'-TGGGCTTAAAACCCACCACT-3') and MG-PARC-B (5'-CGGGTTTCTGTGTAACGCAT-3'). PCR and sequencing of the PCR products were performed as described previously.4

In one man, who had persistent NGU after treatment, pre-treatment M. genitalium had no mutations in the QRDR of the gyrA gene and had a G->T transition at nucleotide position 259 (Asp-87->Tyr) in the parC gene (TableGo). The sequences of the gyrA and parC genes in the post-treatment M. genitalium were the same as those of the pre-treatment mycoplasma. In another man, who remained positive for M. genitalium after treatment despite alleviation of symptoms and no evidence of urethral inflammation, the pre-treatment M. genitalium had no mutations in either the gyrA or parC gene. The post-treatment mycoplasma, however, had a G->A transition at nucleotide position 259 (Asp-87->Asn) in the parC gene (TableGo). This patient did not receive antimicrobial agents after completing 14 days of levofloxacin treatment. Four days later, however, he returned to the clinic with recurrent symptoms. At the time of recurrence of NGU, his first-pass urine was positive for M. genitalium, and the mycoplasma that was detected in the urine also harboured an Asp-87->Asn change in ParC. In the remaining four men, no alterations in GyrA or ParC were found in either the pre- or post-treatment M. genitalium (TableGo).


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Table. Alterations in GyrA and ParC in M. genitalium detected in the first-pass urine of men with NGU before and after 14 days of levofloxacin treatment
 
In this study, we found an amino acid change in ParC of M. genitalium that was detected in the first-pass urine of a patient with NGU before fluoroquinolone treatment. We also observed an amino acid change in ParC of the mycoplasma after levofloxacin treatment. We did not isolate the strains; therefore, we were unable to determine their susceptibilities to fluoroquinolones and to confirm the association of the alterations in ParC with fluoroquinolone resistance. However, these amino acid changes in ParC were analogous to those that could give rise to fluoroquinolone-resistance phenotypes in other bacterial species, including Mycoplasma hominis and Ureaplasma urealyticum.5,6 Therefore, M. genitalium strains with the altered ParC may be less susceptible to fluoroquinolones. In addition, our observations indicate that fluoroquinolone treatment could select fluoroquinolone-resistant mutants in vivo.

In four men who were treated with levofloxacin 100 mg tds for 14 days, M. genitalium was present in the first-pass urine after treatment despite alleviation of symptoms and no evidence of inflammation. However, neither pre- nor post-treatment M. genitalium had alterations in GyrA or ParC. Levofloxacin, the l-isomer of ofloxacin, has only moderate in vitro activity against M. genitalium compared with the high anti-mycoplasmal activity of tetracyclines or macrolides.6 A regimen of levofloxacin 100 mg tds for 14 days may be effective for suppressing mycoplasmal growth and achieving clinical improvement, but this regimen may be insufficient for eradicating the mycoplasma.

Acknowledgements

We thank Kyoko Hirata for technical assistance and laboratory analysis. This study was supported in part by a Grant-in-Aid for Scientific Research (no. 13671643) from the Ministry of Education, Science, Sports and Culture, Japan.

Notes

* Corresponding author. Tel +81-58-265-1241; Fax: +81-58-265-9009; E-mail: deguchit{at}cc.gifu-u.ac.jp Back

References

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4 . Deguchi, T., Yasuda, M., Asano, M., Tada, K., Iwata, H., Komeda, H. et al. (1995). DNA gyrase mutations in quinoloneresistant clinical isolates of Neisseria gonorrhoeae. Antimicrobial Agents and Chemotherapy 39, 561–3.[Abstract]

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6 . Bébéar, C. M., Renaudin, H., Charron, A., Gruson, D., Lefrancois, M. & Bébéar, C. (2000). In vitro activity of trovafloxacin compared to those of five antimicrobials against mycoplasmas including Mycoplasma hominis and Ureaplasma urealyticum fluoroquinolone-resistant isolates that have been genetically characterized. Antimicrobial Agents and Chemotherapy 44, 2557–60.[Abstract/Free Full Text]