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Journal of Clinical Microbiology, March 2007, p. 847-850, Vol. 45, No. 3
0095-1137/07/$08.00+0     doi:10.1128/JCM.02056-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Isolation of Mycoplasma genitalium from First-Void Urine Specimens by Coculture with Vero Cells{triangledown}

Ryoichi Hamasuna,1,2* Yukio Osada,2 and Jørgen Skov Jensen1

Mycoplasma Laboratory, Statens Serum Institut, DK-2300 Copenhagen S, Denmark,1 Department of Urology, Miyazaki Medical College, University of Miyazaki, Kiyotake, Miyazaki, Japan2

Received 5 October 2006/ Returned for modification 22 November 2006/ Accepted 5 January 2007

Isolation of Mycoplasma genitalium from clinical specimens remains difficult. We describe an improvement of the Vero cell coculture method in which the growth of M. genitalium was monitored by quantitative real-time PCR. Four new M. genitalium strains were isolated from six first-void urine specimens of male Japanese patients with urethritis. In two of them, only M. genitalium was detected: one also contained Ureaplasma urealyticum, and one contained Chlamydia trachomatis, Neisseria gonorrhoeae, U. urealyticum, and Ureaplasma parvum. In the specimens yielding isolates of M. genitalium, growth was documented by quantitative PCR after two to five passages in Vero cells. The complete isolation procedure from the initial inoculation to completion of single-colony cloning took about 1 year. Isolation of M. genitalium from urine specimens proved to be more difficult than from swab specimens. Due to the cytotoxic effect of urine, a procedure involving washing of the urinary sediment was introduced. Furthermore, prolonged storage of the urine specimens before culture was shown to be detrimental to the success of isolation, as shown by the lack of success in attempts to isolate M. genitalium from mailed urine specimens as well as by simulation experiments. High concentrations of penicillin G and amphotericin B were surprisingly inhibitory to the growth of wild-type M. genitalium strains, but penicillin G at 200 IU/ml and polymyxin B at 500 µg/ml could be used as selective antibiotics to avoid bacterial overgrowth in the Vero cell cultures.

* Corresponding author. Mailing address: Department of Urology, Miyazaki Medical College, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan. Phone: 81 985 85 2968. Fax: 81 985 85 2968. E-mail: hamaryo{at}med.miyazaki-u.ac.jp.

{triangledown} Published ahead of print on 24 January 2007.

Journal of Clinical Microbiology, March 2007, p. 847-850, Vol. 45, No. 3
0095-1137/07/$08.00+0     doi:10.1128/JCM.02056-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Hamasuna, R., Jensen, J. S., Osada, Y. (2009). Antimicrobial Susceptibilities of Mycoplasma genitalium Strains Examined by Broth Dilution and Quantitative PCR. Antimicrob. Agents Chemother. 53: 4938-4939 [Abstract] [Full Text]  
  • Musatovova, O., Baseman, J. B. (2009). Analysis Identifying Common and Distinct Sequences among Texas Clinical Strains of Mycoplasma genitalium. J. Clin. Microbiol. 47: 1469-1475 [Abstract] [Full Text]  
  • Volokhov, D. V., Kong, H., George, J., Anderson, C., Chizhikov, V. E. (2008). Biological Enrichment of Mycoplasma Agents by Cocultivation with Permissive Cell Cultures. Appl. Environ. Microbiol. 74: 5383-5391 [Abstract] [Full Text]  


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