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 microg/ml could be used as selective antibiotics to avoid bacterial overgrowth in the Vero cell cultures.

FIG. 1.

M. genitalium strain R6G grown in Vero cell culture from a urethral swab specimen from a Danish man was added to urine or SP4 mycoplasma medium kept at room temperature or at 4°C. At 1, 2, 4, 7, 14, and 21 days of incubation, cells from 1.9 ml of urine or SP4 mycoplasma medium were harvested and frozen at −80°C. Each specimen was inoculated into Vero cell cultures, and growth was monitored by quantitative PCR. (A and B) R6G in urine at 4°C (A) and room temperature (B). (C) R6G in SP4 medium at room temperature.