Phylogeny-Based Rapid Identification of Mycoplasmas and Ureaplasmas from Urethritis Patients

Abstract

Some strains of mycoplasmas and ureaplasmas (family Mycoplasmataceae) are associated with nongonococcal urethritis (NGU) or other genitourinary infections. We have developed a rapid and reliable method of identifying the presence and prevalence of mycoplasmas and ureaplasmas in men with NGU. This method is based on the amplification of a part of the 16S rRNA gene by PCR and phylogenetic analysis. A portion of the 16S rRNA gene from 15 prototype strains was amplified with a set of common primers, and their nucleotides were sequenced. The nucleotide sequence of the V4 and V5 regions was analyzed by the neighbor-joining method. The 15 prototype strains were grouped into three distinct clusters, allowing us to clearly segregate the strains into distinct lineages. To determine the prevalence of these pathogens among patients with NGU, this protocol was tested with 148 urine samples. Amplifications were observed for 42 samples, and their nucleotide sequences were analyzed along with those of the 15 prototype strains. The phylogenetic tree thus constructed indicated that 15 of the 42 formed a cluster with Mycoplasma genitalium. Among the remaining specimens, 2 formed a cluster with Mycoplasma hominis, 19 with Ureaplasma urealyticum, and 5 with Ureaplasma parvum; the remaining sample contained both M. genitalium and U. urealyticum. This phylogeny-based identification of mycoplasmas and ureaplasmas provides not only a powerful tool for rapid diagnosis but also the basis for etiological studies of these pathogens. (30,31). Three of these species, M. genitalium, U. parvum, and U. urealyticum, are thought to be associated with genitourinary infections (26,29,30). In experimentally infected chimpanzees, M. genitalium has been shown to induce symptomatic genital infections with inflammatory and antibody responses, suggesting that M. genitalium may be a pathogen of nongonococcal urethritis (NGU) (32). Because it has been extremely difficult to isolate, M. genitalium has not been clearly designated as an etiologic agent in NGU. With the development of PCR-based techniques (14, 20), M. genitalium has been detected to a significantly greater extent in symptomatic males than in asymptomatic males, which suggests that M. genitalium is likely to be an etiologic agent in some cases of NGU (8,11,13,18). For ureaplasmas, several lines of evidence have suggested that U. urealyticum also plays an important role in the etiology of male NGU. However, the colonization rates in asymptomatic males make it difficult to reach an unequivocal conclusion with respect to the etiologic role of U. urealyticum (15,26,30). Previously, U. urealyticum has been differentiated into biovars 1 and 2. Biovar 1 is composed of serovars 1, 3, 6, and 14, and biovar 2 is composed of serovars 2, 4, 5, and 7 to 13 (4, 22, 24). In 1998, U. urealyticum biovars 1 and 2 were classified into U. parvum and U. urealyticum, respectively (17). M. hominis has been associated with bacterial vaginosis, pelvic inflammatory disease, postpartum fever, and postabortal fever, as we...

Quantitative Detection of Mycoplasma genitalium from First-Pass Urine of Men with Urethritis and Asymptomatic Men by Real-Time PCR

“…The AMPLICOR STD-1 assay was performed as described in the manufacturer's instructions. The phylogeny-based assay was performed as described in our previous study (25). All urine specimens that were positive for M. genitalium by the phylogeny-based assay were subjected to the TaqMan assay to quantify M. genitalium DNA.…”

Molecular Diagnostics of Mycoplasmas: Perspectives from the Microbiology Standpoint

“…Thus, simultaneous testing of several species-specific or multiplex PCRs to determine all possible pathogenic mycoplasmas associated with a particular clinical entity would be very complicated. Combination of PCR amplification of a given highly conserved target genome sequence with determination of its nucleotide sequences and phylogenetic analysis has been successfully applied for diagnosis and identification of mycoplasmal etiologies in male urethritis cases [Hashimoto et al, 2006;Yoshida et al, 2002].…”

“…According to the laboratory's infrastructure, the most common methods include: a) culture-based isolation/detection/identification/antimicrobial susceptibility profile; b) antigen detection, c) mycoplasmal-specific serologic responses; and, d) PCR and other NATs. [Razin et al, 1998;Talkington & Waites, 2009;Waites et al, 2000;Yoshida et al, 2002].…”

Longitudinal Quantitative Detection by Real-Time PCR of Mycoplasma genitalium in First-Pass Urine of Men with Recurrent Nongonococcal Urethritis

“…The AMPLICOR STD-1 assay was done according to the manufacturer's instructions. The PCR-and phylogeny-based assay, which could detect at least 10 copies of the 16S rRNA gene of M. genitalium, was done as described in our previous report (18). Two patients were positive for both C. trachomatis and M. genitalium, and three were positive only for M. genitalium.…”

“…With successive visits, we reexamined urethral smears and retested urine for pathogens. DNA samples, which had been prepared from the first-pass urine for the phylogeny-based assay and stored at Ϫ70°C, were subjected to the TaqMan assay for quantification of M. genitalium DNA (17,18). Briefly, precipitate from 1 ml of the first-pass urine was obtained by centrifugation and treated with digestion solution containing proteinase K (18).…”