Bacterial loads of Ureaplasma urealyticum contribute to development of urethritis in men

Abstract

Ureaplasma urealyticum could be a pathogen of non-gonococcal urethritis (NGU) in men. However, ureaplasma is often detected in men without NGU, and the proportion of cases possibly attributable to this pathogen is still undefined. We attempted to determine the bacterial loads of U. urealyticum significantly associated with NGU. The 16S rRNA genes of U. urealyticum were quantified by a real-time polymerase chain reaction-based assay in first-void urine (FVU) from 26 asymptomatic and 25 symptomatic men positive for U. urealyticum. The leucocyte counts in first-void urine (FVU) were determined as an objective measure of inflammatory response to ureaplasma in the hosts by automated quantitative urine particle analysis. Positive correlations were observed between copies of the 16S rRNA genes of U. urealyticum per ml and the leucocyte counts per µl in FVU (r = 0.49, p = 0.0003). Loads of ≥10⁴ copies of the 16S rRNA gene of U. urealyticum/ml, corresponding to ≥5 × 10³ cells of U. urealyticum/ml in FVU, were significantly associated with the presence of urethritis symptoms (p < 0.0001) and with higher leukocyte counts in FVU (p < 0.0001). The bacterial load of U. urealyticum, possibly of ≥5 × 10³ cells of U. urealyticum/ml in FVU, could be significantly associated with the development of symptomatic NGU.

Keywords

Sexually transmitted infection non-gonococcal urethritis bacterial load leukocyte count real-time polymerase chain reaction

Introduction

The tiny-strain mycoplasma isolated from men with non-gonococcal urethritis (NGU) by Shepard et al. in 1954¹ was named Ureaplasma urealyticum in 1974.² Since then, it has been suggested that this ureaplasma might be associated with NGU.³ However, controversy exists regarding the pathogenicity of U. urealyticum in men with NGU because the ureaplasma was frequently detected in the urethras of men without NGU.⁴

Later, U. urealyticum was classified as biovar 1 (parvo biovar) and biovar 2 (T960 biovar).⁵ Biovar 1 became a new independent bacterial species defined as Ureaplasma parvum in 1999,⁶ whereas biovar 2 was defined as U. urealyticum. Our studies on prevalences of these ureaplasmas in male NGU suggested that U. urealyticum (biovar 2) might be associated with NGU but that U. parvum (biovar 1) might colonise the male urethra without causing NGU.^7,8 In other recent studies, the first-void urine (FVU) or urethral swabs from men with and without urethritis symptoms have been examined for the presence of U. urealyticum by nucleic acid amplification tests, and most of the studies have suggested that U. urealyticum might be a pathogen of NGU.^9–12 However, ureaplasma is still often detected in men without urethritis. Such studies focusing only on the prevalence of U. urealyticum in men with and without NGU could not define the proportion of cases possibly attributable to this pathogen.

In earlier studies, in which the two ureaplasma species of U. parvum and U. urealyticum were not distinguished from each other, the association of the bacterial loads of the ureaplasmas with the development of NGU were examined, and the results were controversial.^3,13 However, our quantitative study on the ureaplasmas in urine specimens, in which U. parvum and U. urealyticum were distinguished from each other, showed that the bacterial loads of U. urealyticum were significantly higher in men with NGU than in those without urethritis.¹⁴ To extend our investigation to clarify the pathogenic roles of U. urealyticum in NGU, we distinguished the two species of ureaplasmas from each other, quantified the bacterial loads of U. urealyticum in FVU, and determined the bacterial loads significantly associated with host inflammatory responses, as indicated by leucocyte counts in uncentrifuged FVU, in asymptomatic and symptomatic men in the present study.

Methods

Clinical specimens

The present study comprised 479 men with urethritis symptoms and 256 asymptomatic men who visited a urology clinic (iClinic, Sendai, Japan) between April 2006 and February 2008. The men who complained of one or more urethral symptoms (discharge, dysuria, and urethral burning or irritation) were assigned to the group of symptomatic men, and the men with no current urethral symptoms were assigned to the group of asymptomatic men. All the symptomatic men sought the diagnosis and treatment of urethritis, but most of the asymptomatic men asked for screening for sexually transmitted infections (STIs). FVU, which was the first 20–30 ml of the initial flush of urine after not having urinated for at least 2 h, was collected from each of the 735 subjects. In the symptomatic men, urethral smears were examined for the presence or absence of Neisseria gonorrhoeae to choose antibiotics for treatment of urethritis, but the asymptomatic men were not subjected to urethral smear examinations because of the poor acceptability of the procedure. After collection, a portion of each FVU specimen was immediately examined in the clinic for quantitation of leucocytes, which was performed with automated quantitative urine particle analysers (UF-50 or UF-1000i; Sysmex Corporation, Tokyo, Japan) according to manufacturer instructions. The remaining portion of each FVU specimen was sent to a laboratory (Mitsubishi Chemical Medience Corporation, Tokyo, Japan) for evaluation of the microbial aetiology of urethritis. The urine specimens were tested for N. gonorrhoeae and Chlamydia trachomatis by AMPLICOR STD-1 (Roche Diagnostics, Basel, Switzerland) or APTIMA Combo2 (Gen-Probe Incorporated, San Diego, CA, USA) and for Mycoplasma genitalium, Mycoplasma hominis, U. parvum, and U. urealyticum by polymerase chain reaction (PCR)-microtitre hybridization assay with species-specific oligonucleotide probes, as reported previously.¹⁵ With the informed consent of each man, the FVU specimens remaining after leucocyte quantitation and microorganism examination were stored at −70℃ for additional study.

The present retrospective study was approved by the Institutional Review Board of the Graduate School of Medicine, Gifu University, Gifu, Japan (reference number 22–10). From the 735 subjects, 51 men positive only for U. urealyticum and negative for all other examined pathogens were enrolled in this present study. Of these 51 men, 25 (49.0%) complained of at least one of the urethritis symptoms. Demographic and behavioural characteristics of the subjects, including sexual histories, sexual orientation, and prior urethritis, were not obtained from most of the asymptomatic men. HIV serologic status was not examined in most subjects.

Quantitative detection of U. urealyticum in FVU

The DNAs were extracted from the stored FVU specimens and used as template DNAs for quantitative detection of U. urealyticum by real-time PCR.¹⁴ In the present study, we modified the quantitative real-time PCR assay for the ureaplasmas that we had developed previously. In brief, the PCR mixture contained LightCycler TaqMan Master Pre Mix (Roche Applied Science, Mannheim, Germany), 1 µM each of Ure-1S and Ure-1A, 400 nM of Ure-P1, and 5 μl of the template DNA solution in a total volume of 20 μl. Real-time PCR was performed with a LightCycler (Roche Applied Science) under the following conditions as previously reported. The DNA solutions from all standard dilutions and clinical specimens were run simultaneously with controls containing no template DNA. Quantitative analysis was performed by the automatic second derivative maximum method. Because U. urealyticum has two rRNA operons, the numbers of copies of the 16S rRNA gene detected in FVU corresponded to double the numbers of cells of U. urealyticum existing in FVU.¹⁶

Statistical analysis

The Wilcoxon rank-sum test was used for comparison of differences in distributions of bacterial loads and leucocytes in FVU. Spearman’s rank correlation coefficient was determined between the copies of the 16S rRNA genes of the ureaplasmas and the leucocytes in FVU. Fisher's exact test was used to calculate an exact probability value for the relation between two dichotomous variables. All statistical analyses were two-tailed and were performed with significance set at p < 0.05. Statistical analyses were performed with JMP Version 5.0.1J (SAS Institute Japan, Tokyo, Japan).

Results

Analysed subjects

Of the 479 symptomatic men, 169 (35.3%), 172 (35.9%), 51 (10.6%), 17 (3.5%), 23 (4.8%), and 69 (14.4%) were positive for N. gonorrhoeae, C. trachomatis, M. genitalium, M. hominis, U. parvum, and U. urealyticum, respectively. In the 256 asymptomatic men, 2 (0.8%), 57 (22.3%), 7 (2.7%), 33 (12.9%), 56 (21.9%), and 57 (22.3%) were positive for N. gonorrhoeae, C. trachomatis, M. genitalium, M. hominis, U. parvum, and U. urealyticum, respectively. Some subjects were positive for more than one bacterial species, and five men were coinfected with U. ureaplasma and U. parvum.

Quantitation of leucocytes in FVU

In the men positive for U. urealyticum, leucocyte counts in the FVU specimens ranged from 1.2 to 159.0 leucocytes/µl (median, 4.25 leucocytes/µl) in the 26 asymptomatic men and from 4.5 to 3279.0 leucocytes/µl (median, 157.2 leucocytes/µl) in the 25 symptomatic men. The symptomatic men had significantly higher leucocyte counts in FVU than did the asymptomatic men (p < 0.0001).

Quantitation of U. urealyticum in FVU

The loads of U. urealyticum in the FVU specimens estimated as the copies of the 16S rRNA gene of U. urealyticum ranged from 13 to 97,900 copies/ml (median, 1825 copies/ml) in the 26 asymptomatic and from 137 to 1,860,000 copies/ml (median, 23,900 copies/ml) in the 25 symptomatic men. The symptomatic men had significantly higher loads of U. urealyticum in FVU than did the asymptomatic men (p < 0.0001).

Correlation of bacterial loads of U. urealyticum with leucocyte counts in FVU

Correlations between loads of the 16S rRNA gene of U. urealyticum and leucocyte counts in the FVU in the men positive for U. urealyticum are shown in Figure 1. A positive correlation was found (r = 0.49, p = 0.0003). As the bacterial loads increased, inflammatory responses developed more intensely.

Figure 1.

Correlation of loads of the 16S rRNA gene of U. urealyticum with leucocyte counts in first-void urine from symptomatic (•) and asymptomatic (○) men.

Presumptive cut-off values of bacterial loads of U. urealyticum

With the presumptive cut-off values of loads set at 10³, 10⁴, and 10⁵ copies of the 16S rRNA gene of U. urealyticum per ml of FVU, the differences in leucocyte counts in the FVU specimens between the subjects with less than the cut-off values and those with the cut-off values or greater are shown in Figure 2. At these cut-off values, the associations of the loads with the presence or absence of urethritis symptoms are shown in Table 1. The load of 10⁴ copies of the 16S rRNA gene of U. urealyticum/ml was the most powerful cut-off value showing a significant difference in leucocyte counts in FVU and for distinguishing the presence from the absence of urethritis symptoms.

Figure 2.

Leucocyte counts in first-void urine between the subjects with less than the presumptive cut-off values of loads set at 10³ (a), 10⁴ (b), and 10⁵ (c) copies of the 16S rRNA gene of U. urealyticum per ml of first-void urine and those with the cut-off values or greater, including symptomatic (•) and asymptomatic (○) men. For each box plot, the ends of the rectangle indicate the upper and lower quartiles, the middle line of the box indicates the median, and the tips of the projecting bars show the locations of the minimum and maximum values within the inner fences.

Table 1.

Association of bacterial loads of the 16S rRNA gene of U. urealyticum with the presence or absence of urethritis symptoms.

Bacterial loads (U. urealyticum 16S rRNA gene copies/ml)	No. of men		p value
Bacterial loads (U. urealyticum 16S rRNA gene copies/ml)	Asymptomatic	Symptomatic	p value
<10³	6	2	0.2485
≥10³	20	23
<10⁴	24	7	<0.0001
≥10⁴	2	18
<10⁵	26	20	0.0226
≥10⁵	0	5

Discussion

In our previous quantitative study on the ureaplasmas in FVU, a diagnosis of urethritis was based on the presence of five or more leucocytes per high-power microscopic field (HPF, ×1000) of a Gram-stained urethral smear in men with symptoms compatible with acute urethritis.¹⁴ Men screened for STIs who had no symptoms or signs of urethritis and who showed no more than four leucocytes/HPF in each urethral smear were defined as men without urethritis. We compared the bacterial loads of ureaplasmas in men diagnosed as having definite urethritis and no urethritis. Microscopic observation of urethral smears and urinary sediments is still valuable in estimating inflammation in the urethra for the diagnosis of urethritis but is of limited precision. In the present study, in which we enrolled subjects different from those in our previous study,¹⁴ we examined FVU for quantitation of leucocytes by use of automated analysers with flow cytometry. We adopted the leucocyte counts in FVU as an objective measure of inflammatory response to the ureaplasma in the hosts regardless of the presence or absence of urethritis symptoms¹⁷ and investigated the correlation of the bacterial loads of the ureaplasma with the leucocyte counts in FVU. In the present study, we observed a positive correlation of the loads of U. urealyticum with leucocyte counts in FVU. Loads of ≥10⁴ copies of the 16S rRNA gene of U. urealyticum/ml, corresponding to those of ≥5 × 10³ cells of the ureaplasma/ml, were significantly associated with the presence of urethritis symptoms and with the higher leucocyte counts. Thus, a bacterial load of ≥5 × 10³ cells of U. urealyticum could contribute to the development of symptomatic NGU in men.

The ambiguity of the pathogenic role of U. urealyticum in NGU could be due to several possibilities, one of which might be its low intrinsic virulence in the male urethra. The present study suggests another possibility, that in men who are positive for U. urealyticum but have neither urethritis symptoms nor leucocytes in urethral smears or in urinary sediments, the bacterial load of the ureaplasma might not reach the point at which an inflammatory response develops in the urethra.

In conclusion, the bacterial load of U. urealyticum in the male urethra might be important in the development of inflammatory responses. When the urethra is exposed to a high bacterial load of U. urealyticum, the ureaplasma could cause symptomatic NGU in men. We are fully aware that the limitations of the present study include the small number of subjects and the lack of information regarding sexual behavioural characteristics and HIV serologic status, which might influence the prevalence of ureaplasma infections and the inflammatory response to ureaplasmas in the hosts,^3,18–20 and the absence of detection of ureaplasma subtypes or serovars, which might be associated with pathogenicity of the ureaplasmas.^21,22 Nevertheless, this study is the first report, to our knowledge, to determine the bacterial loads of U. urealyticum significantly associated with the development of NGU. Further studies are needed to definitively establish the pathologic roles of the ureaplasmas in the urogenital tract, and quantitative detection of the ureaplasmas will be indispensable for such research.

Footnotes

Conflict of interest

The authors declare no conflict of interest.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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