TINY (T)-STRAIN MYCOPLASMAS were discovered in human urogenital tract samples in 19541 and were ascribed in 19742 to a new genus and species, Ureaplasma urealyticum. U. urealyticum was divided into 2 biotypes, biovar 1 and biovar 2, and then renamed parvo biovar and T960 biovar, respectively.3 In 1999, on the basis of phylogenetic analysis, strains classified as biovar 1 were designated as a separate species, Ureaplasma parvum, whereas strains with biovar 2 traits retained the U. urealyticum designation.4
Earlier studies in which U. parvum (biovar 1) and U. urealyticum (biovar 2) were distinguished from each other suggested a pathogenic role for Ureaplasma in nongonococcal urethritis (NGU).5 Other studies, however, found that Ureaplasma were frequently isolated from the urethras of healthy men and that there was no significant difference in the prevalence of these Ureaplasma between men with NGU and asymptomatic men.5 Recently, Povlsen et al6 reported that U. urealyticum (biovar 2) was found more often in men with NGU than in men without urethritis. We also reported previously that U. urealyticum (biovar 2) was significantly associated with NGU and with nonchlamydial NGU (NCNGU) and that U. parvum (biovar 1) did not appear to play a pathogenic role in NGU.7 Most recently, however, it was reported that U. parvum (biovar 1) and U. urealyticum (biovar 2) were not associated with NGU.8 Thus, the pathogenicity of Ureaplasma in men with NGU remains controversial. Further studies are needed to clarify the pathologic role of Ureaplasma in NGU.
We previously reported a possible association between the bacterial load of Mycoplasma genitalium and clinical findings and inflammatory responses in NGU.9 Therefore, a quantitative assessment of Ureaplasma may also provide useful information for understanding the pathogenicity of Ureaplasma in the urogenital tract. In the current study, we developed a real-time polymerase chain reaction (PCR) assay to quantify the DNA of Ureaplasma in first-void urine from men with urethritis and men without urethritis and assessed whether the bacterial load of Ureaplasma might be associated with the pathogenicity of U. parvum (biovar 1) or U. urealyticum (biovar 2) in the urogenital tract.
Materials and Methods
Strains and Plasmids
Reference strains of U. urealyticum (T-strain 960), M. genitalium (G37), and Mycoplasma hominis (PG21) were obtained from the National Institute of Infectious Diseases, Tokyo, Japan. The following species of bacteria were purchased from the American Type Culture Collection, Manassas, Virginia: Proteus mirabilis, Pseudomonas aeruginosa, Citrobacter freundii, Streptococcus agalactiae, Morganella morganii, Bacteroides fragilis, Enterobacter cloacae, Staphylococcus epidermidis, Serratia marcescens, Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Staphylococcus aureus, Mycobacterium tuberculosis, U. parvum, Chlamydia trachomatis, and Neisseria gonorrhoeae.
A plasmid harboring a partial sequence of the 16S ribosomal RNA gene of U. urealyticum was prepared as described previously.10 The plasmid solution was adjusted to contain 108 copies/mL of the 16S rRNA gene of U. urealyticum. This stock solution was used to prepare serial 10-fold dilutions ranging from 107 to 103 copies/mL. Bacterial DNA was extracted as described previously.10
Patients and Clinical Samples
Clinical samples were collected from men with urethritis and men without urethritis in the Department of Urology of Toyota Memorial Hospital (Toyota, Japan) between November 1999 and December 2002.7,10,11 A diagnosis of urethritis was based on the presence of 5 or more polymorphonuclear leukocytes per oil-immersion field (×1,000) of a Gram-stained endourethral swab specimen. Men screened for sexually transmitted disease who had no symptoms or signs of urethritis and who showed no more than 4 polymorphonuclear leukocytes per high-power microscopic field in each urethral smear were defined as men without urethritis. First-void urine was obtained from each subject. A portion of the urine was subjected to AMPLICOR STD-1 assay (Roche Diagnostics Systems, Indianapolis, IN) to detect C. trachomatis and N. gonorrhoeae. DNA was prepared from another portion of the urine as described previously.10 In brief, precipitates from 1 mL of urine were harvested by centrifugation at 15,000 g for 30 minutes. Precipitates were then treated with proteinase K at 50°C for 2 hours in digestion buffer, and DNA was extracted with the phenol–chloroform method. After ethanol precipitation, DNA was collected by centrifugation and then resuspended in 50 μL of TE buffer. Ten microliters of each DNA solution was subjected to PCR and phylogeny-based assay for identification of U. parvum (biovar 1), U. urealyticum (biovar 2), M. genitalium, and M. hominis as described previously.10 The remaining DNA was stored at −70°C.
Only samples that were positive for U. parvum (biovar 1) or U. urealyticum (biovar 2) and negative for all other pathogens were included in this study. We collected samples from 14 men with U. parvum (biovar 1)-positive nonmycoplasmal nonchlamydial NGU (NMNCNGU), 23 men with U. urealyticum (biovar 2)-positive NMNCNGU, 20 U. parvum (biovar 1)-positive men without urethritis, and 10 U. urealyticum (biovar 2)-positive men without urethritis.7,10,11 The DNA solutions, which had been stored at −70 °C, were thawed, and 10-μL portions were used for quantification of Ureaplasma bacterial loads.
Quantitative Real-Time Polymerase Chain Reaction
Quantitative real-time PCR was performed with a TaqMan probe. To detect both U. parvum (biovar 1) and U. urealyticum (biovar 2) DNA with equal sensitivity, we designed primers and a probe specific for conserved regions of the 16S rRNA gene of human Ureaplasma.12 The sequence of the forward primer, Ure-1S, was 5′-CTAGATGCTTAACGTCTAGCTGTATCAA-3′ and that of the reverse primer, Ure-1A, was 5′-GCCGACATTTAATGATGATCGT-3′. The sequence of the TaqMan probe, Ure-P1, was 5′-(FAM)-AAGGCGCCAACTTGGACTATCACTGAC-(TAMRA)p-3′. The PCR mixture contained 1× TaqMan buffer A (Applied Biosystems, Foster City, CA); 5.0 mmol/L MgCl2; 0.2 mmol/L each of dATP, dCTP, and dGTP; 0.4 mmol/L dUTP; 500 nM each of Ure-1S and Ure-1A; 200 nM Ure-P1; 1.25 units of AmpliTaq Gold DNA polymerase (Applied Biosystems); 0.5 units of AmpErase (uracil N-glycosylase [UNG]) (Applied Biosystems); and 10 μL template DNA solution in a total volume of 50 μL. Real-time PCR was performed with a Prism 7700 sequence detection system (Applied Biosystems) with the following conditions: UNG was activated for 2 minutes at 50°C followed by deactivation of UNG and subsequent activation of the AmpliTaq Gold DNA polymerase for 10 minutes at 95°C. PCR amplification consisted of 50 cycles of 15 seconds of denaturation at 95°C and 1 minute of annealing and extension at 60°C.
The DNA solutions prepared from the bacterial species, including U. parvum (biovar 1) and U. urealyticum (biovar 2), were tested with this real-time PCR assay. The DNA solutions from clinical specimens and all the standard dilutions were run simultaneously with controls containing no template DNA. From cycle to cycle, ΔRn was calculated by subtracting the baseline fluorescence from the reporter fluorescence normalized by an internal reference. The threshold cycle (Ct) was defined as the cycle number at which the reporter fluorescence exceeded the threshold value, a parameter defined as 10 standard deviations above baseline fluorescence. The logarithm to the base 10 of the initial number of target DNA was proportional to the Ct and could be measured with a standard curve.13 When the copy numbers determined by the assay exceeded the upper limit of the working range, DNA samples were diluted with TE buffer and then assayed again.
The logarithms of copy numbers of the Ureaplasma 16S rRNA gene per 1 mL of first-void urine were calculated and analyzed by Student t test. All statistical analyses were 2-tailed and were performed with significance set at P <0.05.
Genomic DNA from bacterial species, including M. genitalium, M. hominis, U. parvum (biovar 1), and U. urealyticum (biovar 2), was tested with the real-time PCR assay to asses the specificity of the assay. PCR products were obtained only with U. parvum (biovar 1) and U. urealyticum (biovar 2) DNA. No PCR products were detected with DNA of other bacterial species.
We then tested the sensitivity of the real-time PCR assay. Standard dilutions ranging from 105 to 10 copies per reaction of a plasmid containing the 16S rRNA gene of U. urealyticum were amplified by real-time PCR. When the Cts were plotted against the log10 of the initial number of copies of the U. urealyticum 16S rRNA gene, linearity was observed over the range of 105 to 10 copies/reaction (Fig. 1). In addition, a significant coefficient of correlation was obtained repeatedly for the Cts and copy numbers (r >0.990). The lower limit of the assay was 10 copies per reaction. For quantification of U. parvum (biovar 1) and U. urealyticum (biovar 2) in first-void urine specimens, one fifth of the DNA isolated from 1 mL of urine was used for the assay. This yielded a working range of the assay of 5.0 × 105 to 5.0 × 10 copies of U. parvum (biovar 1) or U. urealyticum (biovar 2) DNA per 1 mL of urine.
Thirty-four U. parvum (biovar 1)-positive specimens were subjected to the assay. Of 14 men with NMNCNGU, 11 had bacterial loads of U. parvum (biovar 1) ranging from 7.0 × 106 to 6.2 × 10 copies/mL, and the loads in the remaining 3 men were less than 5.0 × 10 copies/mL. In 20 men without urethritis, U. parvum (biovar 1) loads were below the limit of detection in 5 men, and the loads in the remaining 15 men ranged from 4.1 × 104 to 8.7 × 10 copies/ml (Fig. 2A). U. parvum (biovar 1) was detected at concentrations greater than or equal to 103 copies/mL in 7 (50%) of 14 men with NMNCNGU and in 10 (50%) of 20 men without urethritis.
DNA from 33 U. urealyticum (biovar 2)-positive specimens was also tested with the real-time PCR assay. Of 23 men with NMNCNGU, 21 had bacterial loads of U. urealyticum (biovar 2) in urine ranging from 1.8 × 105 to 5.5 × 10 copies/mL, and the loads in the remaining 2 men were less than 5.0 × 10 copies/mL. Of 10 men without urethritis, 7 had bacterial loads of U. urealyticum (biovar 2) ranging from 4.8 × 103 to 5.7 × 10 copies/mL, and the loads in the remaining 3 men were less than 5.0 × 10 copies/mL (Fig. 2B). U. urealyticum (biovar 2) was detected at concentrations greater than or equal to 103 copies/mL in specimens from 15 (65%) of 23 men with NMNCNGU and one (10%) of 10 men without urethritis.
After the logarithms to the base 10 of copy numbers of the Ureaplasma 16S rRNA gene per 1 mL of first-void urine were calculated, the loads of U. parvum (biovar 1) (means ± standard deviation) in men with NMNCNGU and in men without urethritis were 3.091 ± 1.580 and 2.816 ± 1.113, respectively. U. urealyticum (biovar 2) loads in men with NMNCNGU and in men without urethritis were 3.551 ± 1.260 and 2.162 ± 0.736, respectively. U. parvum (biovar 1) loads did not differ significantly between men with NMNCNGU and men without urethritis (P = 0.556). Men with NMNCNGU had significantly higher U. urealyticum loads in their first-void urine specimens than did the men without urethritis (P <0.005). The difference (95% confidence interval) between U. parvum (biovar 1) loads in men with NMNCNGU and in men without urethritis was 0.2744 (−0.6647 to 1.2134), whereas that between U. urealyticum (biovar 2) loads in men with NMNCNGU and in men without urethritis was 1.3892 (0.5137 to 2.2648). When the probability of a type I error, α, was 0.05 and the difference between the means of Ureaplasma loads in men with NMNCNGU and in men without urethritis was assumed to be 1.5, the statistical power to detect difference was 88.3% and 92.2% for U. parvum (biovar 1) and U. urealyticum (biovar 2), respectively.
In our previous studies, we developed a quantitative real-time PCR assay for detection of M. genitalium and showed that M. genitalium loads were significantly higher in men with acute NCNGU than in asymptomatic men and that the increase in M. genitalium load was associated with emergence of symptoms and signs of NGU in men with chronic urethritis.9,14 In subsequent studies, M. genitalium DNA loads in urethral swab and first-void urine samples were also correlated with symptoms and signs of acute urethritis.15,16 These quantitative findings have strengthened the proposition that M. genitalium causes NGU.
For Ureaplasma, the associations of bacterial loads in the urethra with clinical findings and inflammatory responses in the urethra have been analyzed. In an earlier study, in which 2 species of Ureaplasma were not distinguished from each other, Bowie et al17 detected Ureaplasma by quantitative culture and reported that Ureaplasma were isolated in significantly higher concentrations from first-void urine samples from chlamydia-negative patients with NGU than from subjects without urethritis. In another study, the intensity of clinical symptoms and the numbers of polymorphonuclear leukocytes in urethral smears tended to correlate with the bacterial loads of Ureaplasma in the urethra of men with NGU.18 In contrast, high concentrations of Ureaplasma in first-void urine were also reported in men without urethritis, and there was no significant correlation between the number of leukocytes in the urinary sediment and Ureaplasma loads in the corresponding urine specimens.19 These findings as well as the controversial results of many studies on the prevalence of Ureaplasma in men with urethritis and in men without urethritis have complicated our understanding of the pathogenicity of Ureaplasma in men with NGU.20 In the current study, in which the 2 species were detected individually, higher loads of U. urealyticum (biovar 2) were found to be associated with inflammatory responses in the urethra accompanied by clinical symptoms and signs of acute urethritis. In contrast, the bacterial loads of U. parvum (biovar 1) were not associated with urethritis.
In the current study, the samples chosen from those collected in our previous study7 were examined. In our previous study, U. parvum (biovar 1) was detected in 8.5% of men with NGU and in 13.5% of men without urethritis. U. urealyticum (biovar 2) was detected in 15.8% of men with NGU and in 7.8% of men without urethritis.7 In some cases of NGU, Ureaplasma was detected in various combinations with C. trachomatis, M. genitalium, or M. hominis, although the coexistence of U. parvum (biovar 1) and U. urealyticum (biovar 2) was not observed either in men with urethritis or in men without urethritis. In men who were positive for either of the Ureaplasma but negative for all other pathogens, U. parvum (biovar 1) was detected in 12.6% of men with NMNCNGU and in 13.7% of men without urethritis. U. urealyticum (biovar 2) was detected in 19.7% of men with NMNCNGU and in 7.6% of men without urethritis.7 The prevalence of each Ureaplasma species in men with urethritis and men without urethritis suggested a significant association between U. urealyticum (biovar 2) and NGU.7 Our findings also suggested that the presence of U. parvum (biovar 1) in the male urethra might be the result of colonization and was, therefore, unlikely to contribute significantly to the development of NGU.7 We found a possible association of higher U. urealyticum (biovar 2) loads with symptomatic urethritis and no association of U. parvum (biovar 1) loads with urethritis. These findings support our previous proposition that U. urealyticum (biovar 2) is a pathogen of NGU but that U. parvum (biovar 1) does not cause NGU.
Further studies should be performed for quantitative detection of U. parvum (biovar 1) or U. urealyticum (biovar 2) in men with various conditions, including postgonococcal urethritis and chronic NGU. Such quantitative assessment of Ureaplasma loads in the urethra would be helpful for establishing a pathogenic role of Ureaplasma in the urogenital tract.
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