Relationship of Ureaplasma urealyticum Biovar 2 to Nongonococcal Urethritis

The aim of this study was to determine the frequency of Ureaplasma urealyticum biovars 1 and 2 among 340 men with or without nongonococcal urethritis (NGU) attending a venereal disease clinic in Sweden. NGU was defined by the presence of at least four polymorphonuclear leukocytes per microscopic field (×1,000 magnification) on a smear in which Neisseria gonorrhoeae could not be detected. Ureaplasma urealyticum was detected by polymerase chain reaction, and biovar determination was performed directly on the amplicons by liquid hybridization with biovar-specific probes. Patients with NGU were younger, had had more sexual partners, and exhibited symptoms of urethritis more often than patients without NGU. Ureaplasma urealyticum was detected with the same frequency among patients with and among those without NGU. Among patients with NGU, Ureaplasma urealyticum-positive men were more frequently symptomatic than Ureaplasma urealyticum-negative men. Among patients without NGU, Ureaplasma urealyticum-positive men had had more sexual partners than Ureaplasma urealyticum-negative men. Ureaplasma urealyticum biovar 2 was detected more often among patients with NGU than among those without (P=0.012). Logistic regression analysis was performed using detection of biovar 2 as the response variable and the following four variables as explanatory variables: presence or absence of NGU, symptoms of urethritis, number of partners, and age ≤24 years. The only association found was that between Ureaplasma urealyticum biovar 2 and age ≤24 years. More studies should be conducted to determine the possible pathogenic impact of Ureaplasma urealyticum biovar 2. Electronic Publication     

Detection of Ureaplasma urealyticum by PCR and Biovar Determination by Liquid Hybridization

An assay which combines the direct detection of Ureaplasma urealyticum with biovar determination was developed and applied to 618 urogenital specimens. U. urealyticum was detected by inhibitor-controlled PCR. A 429-bp fragment of the urease gene was amplified. The amplicons were labelled with digoxigenin during PCR. Biovar determination was performed by liquid hybridization with biotin-labelled biovar-specific probes, and the hybrids were detected with peroxidase-conjugated sheep anti-digoxigenin immunoglobulin G Fab fragments. Results of PCR and culture for 453 urogenital specimens from women and 105 urethral specimens from men could be compared. Among the specimens from women, 63% were PCR positive as well as culture positive, 0.9% were positive only by PCR, and 4% were positive only by culture. Among the specimens from men, 15% were PCR positive as well as culture positive, 1% were positive only by PCR, and 9% were positive only by culture. By using culture as the reference method, the PCR had a sensitivity of 94% and a specificity of 98% when applied to specimens from women and a sensitivity of 64% and a specificity of 99% when applied to specimens from men. Overall, 80% of the PCR-positive specimens contained biovar 1,13.5% contained biovar 2, and 6.5% contained both biovars.     

Development of a Monoclonal Antibody to a Ureaplasma urealyticum Serotype 9 Antigen

We produced a monoclonal antibody (MAb) to Ureaplasma urealyticum Vancouver, the serotype 9 standard strain. By immunoblotting, this MAb showed a single, 85-kDa band with the homologous serotype and a minor, 100-kDa band with serotype 2 but did not react with any other serotype standard strain. Clinical isolates of U. urealyticum were tested with this MAb and with two sets of polyclonal antisera against the 14 serotype standard strains. The use of MAb 9-2H9 correctly identified certain serotype 9 strains but did not react with wild-type strains lacking the serotype 9 determinant.     

Comparative in vitro effect of 7 quinolones on Ureaplasma urealyticum

Some new quinolones may be used for the treatment of gonococcal urethritis. U. urealyticum is considered as a potential agent of urethritis. This report describes the in vitro antimicrobial activity of seven quinolones against 45 clinical isolates of U. urealyticum. The MIC's geometric mean is (microgram/ml): rosoxacin (1,74), pefloxacin (4,6), oxolinic acid (9), flumequin (12,12), norfloxacin (15,75), nalidixic acid (27). Pipemidic acid is constantly inactive (greater than 128 micrograms/ml). The results of these susceptibility studies provide support for undertaking clinical evaluations of new quinolones against infections with U. urealyticum.     

Antimicrobial susceptibility of Ureaplasma urealyticum.

An antimicrobial susceptibility test, a tow-tube broth dilution and disk elution method for Ureaplasma urealyticum, was modified to incorporate some of the standard procedures followed in traditional antimicrobial testing. The susceptibility pattern of the species was reevaluated by determining the effect of various antimicrobial agents on 21 vaginal isolates. All isolates were inhibited by tetracycline congeners (1 to 6 micrograms/ml) and killed by methenamine mandelate (0.6 mg/ml). All but one isolates were inhibited by erythromycin (0.4 to 3 micrograms/ml). Only eight isolates were inhibited by nalidixic acid (1 to 6 micrograms/ml), and seven were inhibited by nitrofurantoin (20 to 60 micrograms/ml), whereas all isolates were resistant to rifampin (1 microgram/ml) and trimethoprim-sulfamethoxazole (5 micrograms/ml). The in vitro technique described can readily be performed on individual patient isolates before the initiation of antimicrobial therapy.     

Species identification and subtyping of Ureaplasma parvum and Ureaplasma urealyticum using PCR-based assays

There is good evidence that the organism currently known as Ureaplasma urealyticum should be divided into two species-U. parvum (previously U. urealyticum biovar 1) and U. urealyticum (previously U. urealyticum biovar 2). In this study, we designed a series of primers, targeting the 16S rRNA gene and 16S rRNA-23S rRNA intergenic spacer regions, the urease gene subunits, and the 5' ends of the multiple-banded antigen (MBA) genes, to identify and subtype these Ureaplasma species. All of the species-specific primer pairs could distinguish the two species, but only subtype-specific primer pairs targeting the MBA genes could distinguish subtypes within each species. U. parvum was separated into three subtypes, represented by serovars 1, 3/14, and 6. U. urealyticum was also separated into three subtypes by PCR and/or direct sequencing. Subtype 1 consisted of serovars 2, 5, 8, and 9; subtype 2 contained serovars 4, 10, 12, and 13; and subtype 3 contained serovars 7 and 11. A selection of primer pairs was used to identify and subtype 78 clinical ureaplasma isolates from vaginal swabs of pregnant women and to identify and subtype ureaplasmas directly in 185 vaginal swabs in which they had been previously detected. U. parvum was identified in 228 (87%) of 263 isolates or specimens, and U. urealyticum was identified in 50 (19%) (both were present in 6%). Serovars 3/14 (48%) and 1 (43%) were most common among U. parvum isolates, and subtypes 2 (62%) and 1 (34%) were most common among U. urealyticum isolates. This new PCR-based typing system will facilitate future studies of the relationship between individual Ureaplasma species or subtypes and human disease.     

Hemadsorption by colonies of Ureaplasma urealyticum.

Hemadsorption by colonies of Ureaplasma urealyticum and Mycoplasma pneumoniae differed quantitatively and qualitatively. Using standard methodology, few strains of U. urealyticum hemadsorbed; with a modified method, most strains hemadsorbed, indicating a second type of association. Scanning electron microscopy of tannin-osmium-stained preparations showed guinea pig erythrocytes embedded in ureaplasma colonies and craters left when erythrocytes were dislodged.     

Phase variation of the multiple banded protein in Ureaplasma urealyticum and Ureaplasma parvum

Ureaplasma urealyticum and U. parvum are common commensals and, possibly, pathogens of the human urogenital tract. Like other Mycoplasmatales they possess variable surface proteins. The multiple banded (MB) protein shows a striking variability of its molecular weight. This is caused by changes of the number of C-terminal repeating units. In this study, selective pressure was imposed against cytadherence of U. urealyticum and U. parvum. Ureaplasmas were co-incubated with either erythrocytes or HeLa cells and the cell-bound fraction was removed. Additionally, U. urealyticum populations were transferred serially through broth containing specific polyclonal antibodies. Both approaches led to the emergence of escape variants in which no MB protein was detectable. PCR studies with several primers on different parts of the mba gene indicated major differences between wild-type strains and MB-negative escape variants. In experiments with clonal lineages, however, the loss of the MB protein was shown to be reversible. Therefore, it is proposed that the multiple banded proteins of U. urealyticum and U. parvum are subjected to a phase-switching mechanism as it has already been described for several other Mycoplasmatales.     

Urethral infection of chimpanzees by Ureaplasma urealyticum

Two strains of Ureaplasma urealyticum serotype V that had produced urethritis in human volunteers were, after a number of subcultures in artificial media, introduced intra-urethrally into three chimpanzees. One strain given to two chimpanzees rapidly multiplied 1000-fold whereas there was less evidence that organisms of another strain multiplied in a third animal. Over a 14-day period the ureaplasmas persisted in all animals, did not spread to the throat and did not produce an inflammatory response. After this time they were eliminated by tetracycline therapy.     

418例不孕妇女宫颈解脲脲原体和沙眼衣原体检测分析

目的了解解脲脲原体(Uu)与沙眼衣原体(CT)在不孕妇女中的感染率,探讨Uu、CT感染与不孕症的关系.方法对418例不孕妇女及52例正常早期妊娠妇女的宫颈分泌物进行Uu、CT的检测.结果不孕组Uu、CT阳性率为46.4%、17 7%,均显著高于对照组(P＜0.01).继发不孕组Uu阳性率为59.2%,显著高于原发不孕组(P＜0.01);继发不孕组CT阳性率为19.7%,高于原发不孕组,但无显著性意义(P＞0.05).225例Uu或/和CT感染患者经抗感染治疗后3个月内自然妊娠42例(18.7%).结论Uu、CT感染是导致不孕症的重要因素,把Uu、CT列为不孕妇女的常规检测项目,有利于不孕症的诊治.     

Identification ofMycoplasma hominis,Ureaplasma urealyticum,andChlamydia trachomatis by the polymerase chain reaction

A procedure for identifying chlamydia, mycoplasmas, and ureaplasmas by the polymerase chain reaction is described which uses a system of four primers complementary to fragments of the 16S RNA gene, one of which detects all three microorganisms and the other three are each specific for a particular organism. With this primer system, chlamydia, mycoplasmas, and ureaplasmas are detectable in a single reaction. The procedure may be used for routine diagnoses in diagnostic laboratories. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 12, pp. 606–609, December, 1995 Presented by R. V. Petrov, Member of the Russian Academy of Medical Sciences     

Adherence of Ureaplasma urealyticum to human erythrocytes.

Ureaplasma urealyticum (four serotypes and two clinical isolates) were metabolically labeled with radioactive methionine to a high specific activity. Labeling allowed the study of the mechanism of adherence to human erythrocytes. The adherence mechanism was complex and partially mediated by proteinaceous surface components. The binding sites on the erythrocytes were partially sensitive to neuraminidase treatment, and adherence was inhibited by glycophorin and dextran sulfate, indicating recognition of sialyl residues and sulfated compounds.     

荧光定量聚合酶链反应检测解脲支原体感染的临床意义

目的　采用荧光定量聚合酶链反应 (FQ -PCR)技术 ,准确定量检测待检标本中解脲支原体 (UU)的感染状况。方法　采用FQ -PCR技术 ,检测男女生殖道标本 76 5份 ,并同时用常规PCR技术 ,对比检测了 85例。结果　FQ -PCR检测76 5份标本 ,UU -DNA阳性 2 79份 ,阳性率 36 .5 % ,其DNA平均拷贝数为 2 .4× 10 4。 38例阳性患者治疗两周内复查 ,转阴率仅为 4 4 .7% ,治疗 3w后复查 ,转阴率达 76 % (P <0 .0 0 5 )。常规PCR结果重复检测时 ,2 5例阳性中 2例变为阴性 ,而 6 0例阴性中 4例变为阳性。FQ -PCR结果重复时完全吻合。结论　FQ -PCR检测UU ,具有快速、特异性高、定量准确等优点 ,并可为临床评价疗效提供依据。     

Identification of serotype 1-, 3-, and 6-specific antigens of Ureaplasma urealyticum by using monoclonal antibodies.

Little is known about the antigens responsible for serotype specificity in Ureaplasma urealyticum. We produced monoclonal antibodies to U. urealyticum serotypes 1, 3, and 6, the serotypes most commonly found in pregnant women, and analyzed serotype-specific antigens for the three serotypes. Clinical isolates belonging to serotype 1, 3, or 6 were tested in immunoblots with these monoclonal antibodies. The immunoblot patterns of these isolates were, in most cases, different from each other as well as from those of the reference strains, indicating a high rate of antigenic variation among U. urealyticum strains.     

Cultivation of Ureaplasma urealyticum in continuous culture.

Continuous culture of Ureaplasma urealyticum is reported with a steady-state cell biomass of greater than 10(6) cells per ml. Thus, large cell numbers can be easily obtained; in addition, the system provides a powerful means for exploring what nutrients(s) limits the growth yield of this organism. Urea is shown not to be the growth-limiting nutrient in conventional media, although when provided in excess it appears to be completely hydrolyzed.     

Characterization of the macrophage-stimulating activity from Ureaplasma urealyticum

PROBLEM: Intra-amniotic infection is the most common cause of preterm labor. Infections are thought to cause preterm labor by increasing the production of proinflammatory cytokines at the maternal-fetal interface. Experiments with cell culture and animal models have indicated that bacterial lipopolysaccharide (LPS) increases the production of proinflammatory cytokines in reproductive tissues. The majority of intrauterine infections, however, are associated with Ureaplasma urealyticum, which does not contain LPS. Therefore, we performed a series of experiments to understand better the bacterial factor(s) that are responsible for the proinflammatory effects of U. urealyticum. METHOD OF STUDY: U. urealyticum was cultivated in 3-4 L 10B broth, harvested by centrifugation, washed with saline and frozen at -85 degrees C until use. Cells were then extracted with Triton X-114 and the macrophage-stimulating activity (MSA) of the preparations was studied by evaluating their ability to stimulate tumor necrosis factor-alpha production by a monocytic cell line (THP-1 cells). Additional studies involved testing the sensitivity of the detergent extracts to heating, alkaline hydrolysis and proteinase K digestion. Interaction of Triton X-114 extracts with Toll-like receptor (TLR)-2 and TLR-4 was evaluated using cell lines transfected with one of these receptors, CD14 and a reporter gene. RESULTS: Extraction of U. urealyticum with Triton X-114 demonstrated that the MSA preferentially partitioned to the detergent phase. The MSA of the detergent extracts was abrogated by proteinase K digestion or alkaline hydrolysis but only partially inhibited by heating. Further studies suggested that the detergent extracts could activate both TLR-2 and TLR-4. CONCLUSION: These experiments suggest that the MSA of U. urealyticum is lipophilic, sensitive to alkaline hydrolysis and proteinase K digestion, partially sensitive to heating. These properties are consistent with the activity being due to a lipoprotein. Unlike other Mycoplasma species, the MSA of U. urealyticum appears to interact with both TLR-2 and TLR-4. Purification of the molecule(s) that regulate this activity may provide good therapeutic targets for anti-inflammatory strategies to prevent preterm labor caused by intrauterine infection with U. urealyticum.     

Serotyping of Ureaplasma urealyticum by immunoperoxidase assay.

The immunoperoxidase method was applied to the identification of Urea-plasma urealyticum serotypes. The assay used highly diluted antisera and could be run directly on primary plate isolates. It was ideal for detecting and identifying mixed serotypes because stained and unstained colonies could be visualized simultaneously by conventional light microscopy. Antisera run against eight serotypes revealed one-way cross-reactions between serotypes 3 and 5 and antiserum to 2, and between serotype 4 and antiserum to 8, at dilutions of less than 1:150. This cross-reactivity could be diluted out at the optimal antiserum dilution for the immunoperoxidase assay, but not for the growth inhibition assay or immunofluorescence test. The immunoperoxidase assay therefore proved ideal for serotyping U. urealyticum.     

Mycoplasma hominis and Ureaplasma urealyticum in different gynecologic diseases

The incidence of Mycoplasma hominis (M. hominis) y Ureaplasma urealyticum (U. urealyticum) was investigated in 113 endocervical samples obtained from women who were seen for different gynecological pathologies. Forty-seven (42%) patients were positive to these microorganisms; 26 cases (23%) were positive for M. hominis and 21 (19%; p = NS) for U. urealyticum. Average age was 32.1 +/- 7.7 years; the average number of sexual partners was 1.7 +/- 1.1. Eleven of 17 patients with 3 o more sexual partners were positive for Genital Mycoplasma (GM), and U. urealyticum was found more often in this group. A higher incidence of GM was found in women between 26 and 30 years (34%); 57.5% of the patients with positive cultures for GM had begun sexual activity before 20 years of age. M. Hominis was found in 61% of women with no parity and U. urealyticum in 71% of parous women. The cultures were positive in 10 of 14 patients with pelvic inflammatory diseases (PID). A cervical biopsy was taken from 52 cases and the diagnosis of cervical intraepithelial neoplasia (CIN) was made in 49 (94%) but only 24 of them were positive for GM (50%). Thirty-five patients suffered sterility, and 12 (34%) were positive for GM, however all positive cases consulted because of primary sterility. The conclusions obtained from this study are: 1) Near half of the patients was positive for GM and none of the species was predominant over the other. 2) The more sexual partners the higher was the incidence of GM, especially U. Urealyticum. 3) The lower the age of the first sexual intercourse the higher the probability of contamination with these microorganisms. 4) M. hominis was more common in nulliparous women and U. urealyticum was found more often in parous patients; the number of deliveries did not have influence in these findings. 5) A statistical significance between GM and PID was found (p = 0.03). 6) GM have no influence on spontaneous abortion. 7) No statistical significance was found between GM and the beginning and evolution of CIN. 8) No relation statistically significative was found between GM and sterility.     

解脲脲原体感染的检测与体外耐药性分析

目的探讨本地区解脲脲原体(Uu)感染及耐药情况,指导临床合理用药。方法对临床送检的531份标本进行Uu培养、计数、鉴定和药敏试验;对775份标本进行荧光定量PCR(FQ-PCR)法和培养法检测Uu。结果FQ-PCR法检出Uu阳性339例,阳性率43.74%。培养法检出Uu206例,阳性率38.79%。Uu对9种抗菌药物的耐药率最高为环丙沙星(CIP)80.10%,最低为交沙霉素(JOS)0.00%,敏感率最高为原始霉素(PRI)98.54%。结论Uu用两种方法检测均可有效检出。培养法能提供药敏结果,更有利于临床治疗。药敏结果提示本地区Uu感染经验用药可选择交沙霉素和强力霉素。