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

Abstract

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 g/ml could be used as selective antibiotics to avoid bacterial overgrowth in the Vero cell cultures.Mycoplasma genitalium is one of the pathogens of male urethritis and is sexually transmissible. Two strains of M. genitalium were originally isolated from urethral swabs of male patients with nongonococcal urethritis by direct inoculation of the swabs into SP4 mycoplasma medium (14). However, other investigators failed to isolate new strains from urogenital tract specimens for about 10 years after the first report. In 1996, Jensen et al. recovered five new M. genitalium strains by using Vero cells for initial cocultivation of M. genitalium (8). However, isolation of M. genitalium from clinical specimens remained difficult and time-consuming.In the recent decade, detection of M. genitalium by PCR in first-void urine (FVU) specimens has been shown to be comparable to PCR on urethral swabs and is easier to collect and painless for patients (2, 6, 9). However, M. genitalium has not previously been isolated in axenic culture from FVU. In this study, we improved the culture method and demonstrated its ability to isolate M. genitalium from FVU.(Part of this work was presented at the 15th Meeting of the Japanese Society for Sexually Transmitted Diseases, 4 to 5 December 2004, Tokyo, Japan.) MATERIALS AND METHODSUrine specimens. FVU specimens were collected from 109 Japanese male patients with urethritis. Approximately 30 ml of the urine was centrifuged at 10,000 ϫ g for 30 min. The pellet was resuspended in 1 ml of the same urine and was divided into 0.25-ml aliquots which were stored at Ϫ80°C. ...

Analysis Identifying Common and Distinct Sequences among Texas Clinical Strains of Mycoplasma genitalium

“…Analysis of a growing number of clinical data uncovered significant interstrain sequence variability. Sequences of the MgPa-13 region represent the largest set of data generated from different clinical strains of M. genitalium (13,14,17).…”

Mycoplasma genitalium: from Chrysalis to Multicolored Butterfly

“…As another approach to diagnosis, Jensen and colleagues used a strategy involving the PCR assay to isolate M. genitalium from urogenital specimens (67,104). Vero cell cultures were inoculated with the specimens, and mycoplasmal growth was monitored by PCR technology; when the latter provided evidence of multiplication, the cell culture material was subcultured onto mycoplasma medium.…”

Experimental Infection of Pig-Tailed Macaques (Macaca nemestrina) with Mycoplasma genitalium

“…In addition, accurate and reliable detection of current M. genitalium infection is challenging, requiring nucleic acid amplification techniques not yet commercially available in the United States, further hampering appropriate treatment. Growth of this fastidious pathogen from infected patients is impractical, as its recovery from clinical specimens requires lengthy coculture with mammalian cell lines, a technique available in only a few specialized laboratories (24,25; P. A. Totten, unpublished).…”