The role of Ureaplasma urealyticum in urinary tract infections. Demonstration of antibodies against ureaplasmas in the urine

This study reports the presence of antibodies against Ureaplasma urealyticum in the urine in 17.2% (85) of 493 patients with chronic pyelonephritis. At the same time in 37 of antibody-positive urine samples evidence was found of cultivated Ureaplasma urealyticum, other bacterial germs were isolated infrequently. These culturally and serologically positive urine samples mostly showed pathologic findings of urine sediments. These results led us to believe in a possible involvement of the Ureaplasmas in keeping inflammation processes in the upper urinary tract.     

Bovine abortion and neonatal death associated with Ureaplasma diversum

Ureaplasma diversum was isolated from the lungs and/or stomach fluid and placentas of five aborted bovine fetuses and four newborn calves. All isolates were serotype D48. Placentitis was observed in all instances in which the placenta was examined. Gross lesions consisted of focal or diffuse reddening of the chorioallantois and amnion and thickening of the amnion. Microscopically there were fibrosis, edema and inflammation of the amnion. Microscopic lesions in the lung consisted of diffuse pneumonitis with thickening of the alveolar walls and in some cases peribronchiolar lymphoid accumulations. Macrophages and granulocytes were present in the alveoli. Inoculation of the vulva of a virgin heifer with one of the isolates from a fetal lung produced hyperemia and profuse purulent discharge with slight granularity.     

Purification of urease from Ureaplasma urealyticum

We have purified urease from the Mollicutes, Ureaplasma urealyticum, using high performance liquid chromatography methods and DEAE-Sephadex chromatography. While only small amounts of material could be utilized in these methods, urease was purified at least 180-fold, yield a major band on SDS-PAGE of 66,000 daltons, a minor band of 64,000 daltons, and several faint bands of lower molecular mass. These results suggest that the 380,000 dalton intact urease is a pentamer or hexamer of these two larger subunits. The highly purified urease from DEAE-Sephadex retained full activity for at least 20 days at 4 degrees C in sodium phosphate buffer (pH 7.2) with 1% bovine serum albumin. The estimated specific activity of the DEAE peak fractions, 180 IU/micrograms, is at least 90-fold greater than that of jack bean urease.     

Characteristics of Ureaplasma urealyticum urease.

Sonication of Ureaplasma urealyticum cells grown in a dialysate growth medium effectively separated the cytoplasmic fraction from the membrane fraction, with both fractions relatively free from exogenous contaminating proteins. The urease activity was associated with the cytoplasmic fraction, and the ureaplasmal urease exhibited a specific activity higher than that of crystalline jack bean urease. The enzymatic activity of the ureaplasmal enzyme was optimum at pH 7.5 and was resistant to the chelating agents EDTA and sodium citrate. Sulfhydryl-blocking agents such as HgCl2 and Pb(NO3)2 inhibited the ureaplasmal urease, which was also shown to be particularly sensitive to flurofamide and, to a much lesser extent, to acetohydroxamic acid. Electrophoretic analysis of the proteins of the ureaplasmal cell fractions combined with Western immunoblot with an antiserum to the ureaplasmal urease indicated that the urease constitutes a major component of the cytoplasm and is composed of several 70-kilodalton polypeptides.     

Apoptosis in HEp-2 cells infected with Ureaplasma diversum

Background

Bacterial pathogens have many strategies for infecting and persisting in host cells. Adhesion, invasion and intracellular life are important features in the biology of mollicutes. The intracellular location of Ureaplasma diversum may trigger disturbances in the host cell. This includes activation or inhibition of pro and anti-apoptotic factors, which facilitate the development of host damage. The aim of the present study was to associate U. diversum infection in HEp-2 cells and apoptosis induction. Cells were infected for 72hs with four U. diversum clinical isolates and an ATCC strain. The U. diversum invasion was analyzed by Confocal Laser Scanning Microscopy and gentamicin invasion assay. The apoptosis was evaluated using pro-apoptotic and anti-apoptotic gene expression, and FITC Annexin V/Dead Cell Apoptosis Kit.

Results

The number of internalized ureaplasma in HEp-2 cells increased significantly throughout the infection. The flow cytometry analysis with fluorochromes to detect membrane depolarization and gene expression for caspase 2, 3 and 9 increased in infected cells after 24 hours. However, after 72 hours a considerable decrease of apoptotic cells was observed.

Conclusions

The data suggests that apoptosis may be initially induced by some isolates in association with HEp-2 cells, but over time, there was no evidence of apoptosis in the presence of ureaplasma and HEp-2 cells. The initial increase and then decrease in apoptosis could be related to bacterial pathogen-associated molecular pattern (PAMPS). Moreover, the isolates of U. diversum presented differences in the studied parameters for apoptosis. It was also observed that the amount of microorganisms was not proportional to the induction of apoptosis in HEp-2 cells.     

Invasion of Ureaplasma diversum in Hep-2 cells

Background

Mozambique is one of the countries with the highest burden of tuberculosis (TB) in Sub-Saharan Africa, and information on the predominant genotypes of Mycobacterium tuberculosis circulating in the country are important to better understand the epidemic. This study determined the predominant strain lineages that cause TB in Mozambique.

Results

A total of 445 M. tuberculosis isolates from seven different provinces of Mozambique were characterized by spoligotyping and resulting profiles were compared with the international spoligotyping database SITVIT2. The four most predominant lineages observed were: the Latin-American Mediterranean (LAM, n = 165 or 37%); the East African-Indian (EAI, n = 132 or 29.7%); an evolutionary recent but yet ill-defined T clade, (n = 52 or 11.6%); and the globally-emerging Beijing clone, (n = 31 or 7%). A high spoligotype diversity was found for the EAI, LAM and T lineages.

Conclusions

The TB epidemic in Mozambique is caused by a wide diversity of spoligotypes with predominance of LAM, EAI, T and Beijing lineages.     

Comparison between Culture and a Multiplex Quantitative Real-Time Polymerase Chain Reaction Assay Detecting Ureaplasma urealyticum and U. parvum

A novel multiplex quantitative real-time polymerase chain reaction (qPCR) for simultaneous detection of U. urealyticum and U. parvum was developed and compared with quantitative culture in Shepard''s 10 C medium for ureaplasmas in urethral swabs from 129 men and 66 women, and cervical swabs from 61 women. Using culture as the gold standard, the sensitivity of the qPCR was 96% and 95% for female urethral and cervical swabs, respectively. In male urethral swabs the sensitivity was 89%. The corresponding specificities were 100%, 87% and 99%. The qPCR showed a linear increasing DNA copy number with increasing colour-changing units. Although slightly less sensitive than culture, this multiplex qPCR assay detecting U. urealyticum and U. parvum constitutes a simple and fast alternative to the traditional methods for identification of ureaplasmas and allows simultaneous species differentiation and quantitation in clinical samples. Furthermore, specimens overgrown by other bacteria using the culture method can be evaluated in the qPCR.     

解脲脲原体感染与不孕不育

解脲脲原体是人类泌尿生殖道常见的寄生菌之一,它可以引起男女泌尿生殖道感染,其中最严重的后果认为可导致男女不孕不育。支原体感染是否与男性和女性的不孕不育有关,其致病的主要机制是什么,探讨这些问题对于防治解脲脲原体感染,减少及避免不孕不育疾病的发生有重要意义。本文对近年来关于解脲脲原体感染导致女性不孕和男性不育的机制进行了综合概述。     

Comparison of two methods for serotyping Ureaplasma urealyticum clinical isolates

A newly developed enzyme linked immunosorbent assay (ELISA) method using monoclonal antibodies (MAbs) to the 14 serotypes of Ureaplasma urealyticum was compared to immunofluorescence assay (IFA) for serotyping U. urealyticum clinical isolates. Of the 102 vaginal isolates of U. urealyticum, five strains were lost and were excluded from analysis. Of the 97 strains analysed, a total of 86 (89%) strains were typeable by ELISA and a total of 89 (92%) strains were typeable by IFA. Eighty-six strains were typeable by both methods, three by IFA only and eight strains were not typeable neither by ELISA nor by IFA. Of the 86 strains typeable by both methods, complete concordance in serotyping results was found. The three strains not typeable by ELISA were typeable as serotype 4 by IFA. These three strains were reanalysed by ELISA after major modifications of the antigen preparation and were typeable as serotype 4. In conclusion, the ELISA was found suitable for serotyping clinical isolates. However, since the ELISA had a somewhat lower performance than IFA, strains not typeable by ELISA, should be retested by another technique such as IFA.     

Cell fractions and enzymatic activities of Ureaplasma urealyticum.

The localization of some enzymic activities in cell fractions of Ureaplasma urealyticum was studied. A quantitative evaluation of the effectiveness of several cell lysis procedures was obtained by using labeled membranes and sucrose density gradient centrifugation. Ultrasonic treatment was found to be the most effective procedure for lysing the cells, whereas digitonin and osmotic shock caused the lysis of only 70 and 50% of the cells, respectively. The localization of selected enzymes in Ureaplasma cells resembled that found in other Mycoplasma species. Adenosine triphosphatase, ribonuclease, deoxyribonuclease, and p-nitrophenylphosphatase activities were located exclusively in the membrane fraction, whereas urease and L-histidine ammonia-lyase were located in the cytoplasm.     

Ureaplasma urealyticum and Ureaplasma parvum in etiology of urogenital mixt-infections

The complex study of 104 vaginal samples from patients with urogenital uroplasmosis was carried out. U. parvum were detected in 67.3% patients, U. urealyticum--in 12.5% and in 20.1% cases--two species were registered at the same time. Isolation of clinical significant concentration of both ureaplasma (> 10(4) CFU/ ml) was detected in about 50% of cases. Expression of inflammation of vaginal mucus depended on the level of concentration of infection agents. U. parvum were associated with bacterial vaginosis, while in urogenital candidosis U. parvum was detected rarer than U. urealyticum. The dominant numbers of clinical ureaplasma were high sensitive to "new" macrolides and chinolons, however the high percent of isolates were resistant to erytromicin and doxiciclin.     

High-density lipoprotein that supports Ureaplasma urealyticum growth.

A high-density lipoprotein with growth-promoting activity for Ureaplasma urealyticum was purified in high yield from equine serum by ammonium sulfate fractionation and molecular filtration. Fractions enriched in growth-promoting activity represented 5% of the total serum protein, and 30 micrograms of the purified protein per ml gave an activity equivalent to that from 100 micrograms of whole serum per ml. The serum was totally replaced by purified lipoprotein when tested in a soy peptone-yeast dialysate or when added to a chemically defined synthetic medium. Polyacrylamide gel electrophoresis indicated that one major protein with growth-promoting activity is present. A total of 10 proteins were distinguished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with 75% of the total contributed by two proteins with molecular weights of 160,000 and 170,000. A total of 90% of the lipoprotein was an alpha-protein with a mobility of 0.67 in two-dimensional immunoelectrophoresis (albumin = 1.0). The active component was further characterized as high-density lipoprotein by density ultracentrifugation. Two components with S = 6.4 and S = 15.8 were distinguished by velocity sedimentation. The lipid was removed from lipoprotein during its precipitation with acetone. The growth-promoting activity of delipidized protein was dependent upon the addition of exogenous cholesterol, and [14C]cholesterol was transferred to urea-plasmic cells in cultures containing the delipidized protein. A major portion of the [14C]cholesterol remained associated with the protein during filtration on Sepharose 4B columns.     

Comparative genome analysis of 19 Ureaplasma urealyticum and Ureaplasma parvum strains

ABSTRACT: BACKGROUND: Ureaplasma urealyticum (UUR) and Ureaplasma parvum (UPA) are sexually transmitted bacteria among humans implicated in a variety of disease states including but not limited to: nongonococcal urethritis, infertility, adverse pregnancy outcomes, chorioamnionitis, and bronchopulmonary dysplasia in neonates. There are 10 distinct serotypes of UUR and 4 of UPA. Efforts to determine whether difference in pathogenic potential exists at the ureaplasma serovar level have been hampered by limitations of antibody-based typing methods, multiple cross-reactions and poor discriminating capacity in clinical samples containing two or more serovars. RESULTS: We determined the genome sequences of the American Type Culture Collection (ATCC) type strains of all UUR and UPA serovars as well as four clinical isolates of UUR for which we were not able to determine serovar designation. UPA serovars had 0.750.78 Mbp genomes and UUR serovars were 0.840.95 Mbp. The original classification of ureaplasma isolates into distinct serovars was largely based on differences in the major ureaplasma surface antigen called the multiple banded antigen (MBA) and reactions of human and animal sera to the organisms. Whole genome analysis of the 14 serovars and the 4 clinical isolates showed the mba gene was part of a large superfamily, which is a phase variable gene system, and that some serovars have identical sets of mba genes. Most of the differences among serovars are hypothetical genes, and in general the two species and 14 serovars are extremely similar at the genome level. CONCLUSIONS: Comparative genome analysis suggests UUR is more capable of acquiring genes horizontally, which may contribute to its greater virulence for some conditions. The 4 overwhelming evidence of extensive horizontal gene transfer among these organisms from our previous studies combined with our comparative analysis indicates that 6 ureaplasmas exist as quasispecies rather than as stable serovars in their native environment. Therefore, differential pathogenicity and clinical outcome of a ureaplasmal infection is most likely not on the serovar level, but rather may be due to the presence or absence of potential pathogenicity factors in an individual ureaplasma clinical isolate and/or patient to patient differences in terms of autoimmunity and microbiome.     

The infection of monkeys with Ureaplasma urealyticum serovar VIII

The possibility of modeling chronic infection on monkeys by the injection of the culture of U. urealyticum, serotype VIII, was shown. The infection of monkeys with these microorganisms introduced in a single intraperitoneal injection resulted in the generalization of the process, which was manifested by the persistence and reproduction of the infective agent in the organs and blood of the animals for as long as 6 months (the term of observation). Lymphoid hyperplasia in the organs of immunogenesis and transitory immunomorphological reaction in the tissues of some organs of the urogenital system were noted. The localization of infective agents in some endocrine glands was not accompanied by disturbances in their function.