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Several species of Ureaplasma bacteria are known to be present in the urogenital tract of humans, in both healthy individuals and symptomatic patients. These pathogens are associated with urogenital tract infections, infertility problems and spontaneous abortion in humans. The present study involved 77 strains of Ureaplasma species (Ureaplasma spp.), including 21 Ureaplasma urealyticum (U. urealyticum) strains and 56 Ureaplasma parvum (U. parvum) strains. Lipoic acid (LA) and its reduced form dihydrolipoic acid (DHLA) are synthesized in all prokaryotic and eukaryotic cells. Research of recent years increasingly points to therapeutic properties of exogenously supplemented LA. In our study, we examined for the first time the effect of LA on the bacteria multiplication and its bactericidal activity against U. urealyticum and U. parvum. The LA concentrations used were: 1200 µg/ml, 120 µg/ml, and 12 µg/ml. The titer for each strain of Ureaplasma spp. was estimated using the color changing units (CCU) assay. For CCU measurements, a series of 10-fold dilutions of each cell culture in 0.9% NaCl (titration) was prepared and 1 CCU/ml was defined as the highest dilution of cells at which color change was detected. The strongest bacteriostatic and bactericidal effect of LA was observed at a concentration of 1200 µg/ml. In contrast, at lower LA concentrations, stimulation of the bacteria multiplication was noted for 14% of the total number of strains tested. Taken together, the current data provide novel findings about potential beneficial antimicrobial effects of LA.

Ureaplasma spp. have gained increasing recognition as pathogens in both adult and neonatal patients with multiple clinical presentations. However, the clonality of this organism in the male population and infertile couples in China is largely unknown. In this study, 96 (53 U. parvum and 43 U. urealyticum) of 103 Ureaplasma spp. strains recovered from genital specimens from male patients and 15 pairs of infertile couples were analyzed using multilocus sequence typing (MLST)/expanded multilocus sequence typing (eMLST) schemes. A total of 39 sequence types (STs) and 53 expanded sequence types (eSTs) were identified, with three predominant STs (ST1, ST9 and ST22) and eSTs (eST16, eST41 and eST82). Moreover, phylogenetic analysis revealed two distinct clusters that were highly congruent with the taxonomic differences between the two Ureaplasma species. We found significant differences in the distributions of both clusters and sub-groups between the male and female patients (P < 0.001). Moreover, 66.7% and 40.0% of the male and female partners of the infertile couples tested positive for Ureaplasma spp. The present study also attained excellent agreement of the identification of both Ureaplasma species between paired urine and semen specimens from the male partners (k > 0.80). However, this concordance was observed only for the detection of U. urealyticum within the infertile couples. In conclusion, the distributions of the clusters and sub-groups significantly differed between the male and female patients. U. urealyticum is more likely to transmit between infertile couples and be associated with clinical manifestations by the specific epidemic clonal lineages.

Antibiotic resistance in Ureaplasma urealyticum/Ureaplasma parvum and Mycoplasma hominis is an issue of increasing importance. However, data regarding the susceptibility and, more importantly, the clonality of these organisms are limited. We analyzed 140 genital samples obtained in Bern, Switzerland, in 2014. Identification and antimicrobial susceptibility tests were performed by using the Mycoplasma IST 2 kit and sequencing of 16S rRNA genes. MICs for ciprofloxacin and azithromycin were obtained in broth microdilution assays. Clonality was analyzed with PCR-based subtyping and multilocus sequence typing (MLST), whereas quinolone resistance and macrolide resistance were studied by sequencing gyrA, gyrB, parC, and parE genes, as well as 23S rRNA genes and genes encoding L4/L22 ribosomal proteins. A total of 103 samples were confirmed as positive for U. urealyticum/U. parvum, whereas 21 were positive for both U. urealyticum/U. parvum and M. hominis. According to the IST 2 kit, the rates of nonsusceptibility were highest for ciprofloxacin (19.4%) and ofloxacin (9.7%), whereas low rates were observed for clarithromycin (4.9%), erythromycin (1.9%), and azithromycin (1%). However, inconsistent results between microdilution and IST 2 kit assays were recorded. Various sequence types (STs) observed previously in China (ST1, ST2, ST4, ST9, ST22, and ST47), as well as eight novel lineages, were detected. Only some quinolone-resistant isolates had amino acid substitutions in ParC (Ser83Leu in U. parvum of serovar 6) and ParE (Val417Thr in U. parvum of serovar 1 and the novel Thr417Val substitution in U. urealyticum). Isolates with mutations in 23S rRNA or substitutions in L4/L22 were not detected. This is the first study analyzing the susceptibility of U. urealyticum/U. parvum isolates in Switzerland and the clonality outside China. Resistance rates were low compared to those in other countries. We hypothesize that some hyperepidemic STs spread worldwide via sexual intercourse. Large combined microbiological and clinical studies should address this important issue., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Aim: Study of preservation dynamics of ureaplasma laboratory strain live cultures and their DNA in transport medium at varying temperature., Materials and Methods: The study was carried out in laboratory strains Ureaplasma urealyticum serotype 8 and Ureaplasma parvum serotype 1. The quantity of live ureaplasmas was determined by method of tenfold dilutions in liquid medium. The growth of ureaplasmas was registered by changes in the color of the cultivation medium due to its alkalization by metabolism products and expressed in CCU/ml. DNA quantity in samples was determined by real time PCR performed by using Florocenosis-micoplasmas-FL test system produced by ILS., Results: Live ureaplasmas wer shown to be preserved in transport medium at 4 degrees C for 12 - 29 days, at 18 - 22 degrees C--for 9 - 20 days and at 37 degrees C--for only 2 days. In samples incubated at 37 degrees C the quantity of live ureaplasmas increased and then sharply decreased to 0, at lower temperature titers of the cells decreased smoothly. The quantity of ureaplasma DNA in the process of their incubation did not change significantly., Conclusion: Fundamental differences in the duration of survival of U. urealyticum strain and U. parvum strain in transport medium at varying temperature were not detected. Based on the studies performed a practical conclusion can be drawn that in cases of emergency when clinical material transportation is necessary its storage in transport medium for several days is acceptable.

Ureaplasma parvum and Ureaplasma urealyticum are sexually transmitted, opportunistic pathogens of the human urogenital tract. There are 14 known serovars distributed between the two species. For decades, it has been postulated based upon limited data that virulence is related to serotype specificity. The results were often inconclusive due to the small sample size and extensive cross-reactivity between certain serovars. We developed real-time quantitative PCRs that allow reliable differentiation of the two species and type strains of each of the 14 serovars. To investigate species and serovar distributions, we typed 1,061 clinical isolates of human ureaplasmas from diverse patient populations. There was only a tenuous association between individual Ureaplasma serovars and certain patient populations. This may in part be explained by the fact that almost 40% of the isolates were genetic mosaics, apparently arising from the recombination of multiple serovars. This explains the extensive cross-reactivity based upon serotyping and the lack of consistent association of given serotypes with disease.

To explore the possibilities of a novel multiplex real-time PCR system for rapid diagnosis, genetic typing of serovars and clinical application in NGU, we developed a multiplex real-time PCR system for the simultaneous diagnosis of Chlamydia trachomatis, Ureaplasma parvum and Ureaplasma urealyticum and molecular detection of serovars of C. trachomatis and U. parvum in NGU using the SNP technology and TaqMan-LNA probe. In 57 pathogen-positive clinical specimens, we identified the following C. trachomatis serovars: D (20.05%, 12/57), E (36.84%, 21/57), F (19.30%, 11/57), G (8.77%, 5/57), H (5.26%, 3/57), J (3.51%, 2/57), and K (5.26%, 3/57). In 115 pathogen-positive clinical specimens, we identified the following U. parvum serovars: 1 (0.87%, 2/115), 3 (55.65%, 64/115), 6 (20.87%, 24/115) and 14 (21.74%, 25/115). Our fast pathogen diagnosis and serotyping assay using real-time TaqMan-LNA PCR may improve our ability to study the pathogenesis and epidemiology of NGU., (Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.)

Ureaplasma urealyticum is a causative agent of non-gonococcal urethritis and is implicated in the pathogenesis of prostatitis, epididymitis and infertility. The organism is more common in partners of infertile than fertile marriages. U. urealyticum infections not only jeopardize fertility but also pose a risk for infertility treatment and resulting pregnancies. The aim of this study was to determine the prevalence of U. urealyticum and Ureaplasma parvum in semen of infertile and healthy men by polymerase chain reaction (PCR). Semen samples were obtained from infertile patients and healthy controls and were subjected to the routine andrological analysis and PCR. DNA was extracted by the Cadieux method, and analysed by PCR protocol with specific primers for urease and multiple-banded antigen genes. Ureaplasmas were detected significantly by PCR in 12 of 100 (12%) semen specimens from infertile patients and in three of 100 (3%) healthy men. The volume of semen fluid, concentration of sperm cells, and sperm cell with normal morphology were significantly decreased in infertile men. In the group of infertile patients with PCR positive for Ureaplasmas, the volume, count and morphology of semen samples were lower than in the infertile patients with PCR-negative results. U. urealyticum species in semen of infertile men was found to be high (9%) than in healthy controls (1%). Detection rate for U. parvum was 3% in the infertile group and 2% in healthy men. The results indicate that U. urealyticum species is more common in specimens of infertile men. The percentage of normal sperm cells, the volume of semen and the percentage of sperm cells with motility in the PCR positive for U. urealyticum species group were lower than in the PCR positive for U. parvum group.

Studies of epidemiology and pathogenesis of infections with Ureaplasma have received much attention in recent years. Rapid molecular methods for serotyping of Ureaplasma parvum will be of great value in the studies. We developed 2 multiplex real-time TaqMan polymerase chain reaction systems that allow simultaneous detecting and serotyping of 4 serovars of U. parvum.

Evidence has been presented that the species currently known as Ureaplasma urealyticum should be separated into 2 species-Ureaplasma parvum (previously, U. urealyticum biovar 1) and U. urealyticum (previously, U. urealyticum biovar 2). Differentiation and quantification of U. parvum and U. urealyticum can provide important information of the epidemiology of Ureaplasma infections. We developed 2 real-time TaqMan polymerase chain reaction (PCR) assays that would allow rapid, specific, sensitive, quantitative detection and convenient differentiation of U. parvum and U. urealyticum. One hundred twenty-eight clinical specimens were studied and compared with results obtained by culture methods and conventional PCR. The positive rate of real-time TaqMan PCR (59.4%, 76 of 128) was higher than that of culture methods (42.2%, 54 of 128) and conventional PCR (50%, 64 of 128). Of 76 positive specimens, 86.8% (66) contained U. parvum only, 10.5% (8) contained U. urealyticum only, and 2.6% (2) contained both. The copy numbers of 11 positive specimens were in the range of 10(1) to 10(3) copies per reaction mixture, 18 in the range of 10(3) to 10(5), and 47 in the range of 10(5) to 10(8). In the future, quantitative detection and convenient differentiation of real-time TaqMan PCR assays will assist in the study of the pathogenesis and epidemiology of Ureaplasma infections.

We describe the first use of pulsed field gel electrophoresis to genotype human Ureaplasma species. This technique can distinguish between U. urealyticum and U. parvum, differentiate most of the 14 serovars from one another, and identify differences among clinical isolates of the same serovar.

Background: Ureaplasma urealyticum and U. parvum have been associated with respiratory diseases in premature newborns, but their role in the pathogenesis of the respiratory distress syndrome (RDS) is unclear. The aim of this study was to detect, using molecular techniques, the role of Mycoplasma spp. and Ureaplasma spp. in respiratory secretion and blood specimens of preterm newborns with or without RDS and to evaluate the prevalence of perinatal U. urealyticum or U. parvum infection. The influence of chemotherapy on the clinical course was also evaluated., Methods: Tracheal aspirate or nasopharingeal fluid samples from 50 preterm babies with (24) or without RDS (26) were analysed for detection of U. urealyticum and U. parvum by culture identification assay and PCR. Sequencing analysis of amplicons allowed us to verify the specificity of methods. Clarithromycin (10 mg kg-1 twice a day) was administered in ureaplasma-positive patients who presented clinical signs of RDS., Results: 15/24 neonates with RDS (p < 0.001) and 4/26 without RDS were found PCR-positive for U. urealyticum or U. parvum. Culture identification assay was positive in 5/50 newborns, three of which with RDS. Sequencing analyses confirmed the specificity of these methods. Association of patent ductus arteriosus with ureaplasma colonization was more statistically significant (p = 0.0004) in patients with RDS than in those without RDS., Conclusion: Colonization of the lower respiratory tract by Ureaplasma spp. and particularly by U. parvum in preterm newborns was related to RDS. The routine use of molecular methods could be useful to screen candidate babies for etiologic therapy.

A PCR assay was used to analyze endotracheal aspirates from preterm infants for Ureaplasma parvum versus U. urealyticum. U. parvum was detected more often than U. urealyticum. There was no significant difference or trend in the prevalence of either species between infants with or without bronchopulmonary dysplasia when isolated alone.

Ureaplasma parvum and Ureaplasma urealyticum, also known as biovar parvum and biovar T960, respectively, could be associated with several disorders in men, women, and mainly, in newborn children with under weight. Several methods have been developed in order to identify the species or biovars of ureaplasmas. We developed a Multiplex-PCR method using the UPS-UPSA and UUS2-UUA2 primers, specific for U. parvum and U. urealyticum, respectively. This Multiplex-PCR method was used to identify cultures of clinical positive samples to Ureaplasma spp. by the "MYCOFAST Evolution-2" Kit. Of 56 positive cultures to Ureaplasma spp. from newborn children, 70% were U. parvum and 30% U. urealyticum; in 76 positive samples in women, 83% corresponded to U. parvum and 17% to U. urealyticum, while in 63 positive samples of men, 76% identified U. parvum and 24% U. urealyticum. The PCR-multiplex method showed specificity for the identification of the biovars or species of ureaplasmas of clinical interest.

The phenotypic and genotypic properties of Ureaplasma urealyticum (family Mycoplasmataceae, order Mycoplasmatales, class Mollicutes) are reviewed here. The 14 recognized serovar standard strains found in humans exhibit no serological cross-reactivity with ureaplasmas from other hosts and uniquely express human immuoglobulin A1 protease activity. However, they exhibit many characteristics which place them in two distinct clusters known as the parvo biovar (or biovar 1 or B) and the T960T biovar (or biovar 2 or A). Established phenotypic markers of the biovars include clustering of antigenic types, polypeptide patterns of whole-cell preparations, differential inhibition by manganese, and polymorphism among their ureases, pyrophosphatases and diaphorases. Established genotypic markers of the biovars are DNA-DNA hybridization of 60% between biovars, and distinctive RFLP patterns and genome sizes. Divergent nucleotide sequences of several highly conserved genes attest to the phylogenetic distinctiveness of the two biovars. PCRs founded upon the sequences for 16S rRNA, the 16S-23S rRNA intergenic regions, the genus-defining urease, the serovar-defining, multiple-banded antigen genes or randomly amplified polymorphic DNA tests differentiate the biovars unambiguously. With the availability of rapid, reliable and economical tests for biovar determination, it is now appropriate to propose that the taxonomic status of U. urealyticum be emended. Serovar standard strains exhibiting traits of biovar parvo (serovars 1, 3, 6 and 14) will be designated as a separate species, Ureaplasma parvum sp. nov., as befits its smaller genome size. The serovar 3 standard (strain 27T) will be the type strain of U. parvum and is represented by ATCC 27815T and NCTC 11736T. Serovar standard strains exhibiting traits of biovar T960T (2, 4, 5, 7, 8T, 9, 10, 11, 12 and 13) will retain the U. urealyticum designation and type strain, the serovar 8 standard (strain T960T), represented by ATCC 27618T and NCTC 10177T.

Ureaplasma urealyticum has been divided into 14 serovars. Recently, subdivision of U. urealyticum into two species has been proposed: U. parvum (previously U. urealyticum parvo biovar), comprising four serovars (1, 3, 6, 14) and U. urealyticum (previously U. urealyticum T-960 biovar), 10 serovars (2, 4, 5, 7-13). The multiple-banded antigen (MBA) genes of these species contain both species and serovar/subtype specific sequences. Based on whole sequences of the 5'-ends of MBA genes of U. parvum serovars and partial sequences of the 5'-ends of MBA genes of U. urealyticum serovars, we previously divided each of these species into three MBA genotypes. To further elucidate the relationships between serovars, we sequenced the whole 5'-ends of MBA genes of all 10 U. urealyticum serovars and partial repetitive regions of these genes from all serovars of U. parvum and U. urealyticum. For the first time, all four serovars of U. parvum were clearly differentiated from each other. In addition, the 10 serovars of U. urealyticum were divided into five MBA genotypes, as follows: MBA genotype A comprises serovars 2, 5, 8; MBA genotype B, serovar 10 only; MBA genotype C, serovars 4, 12, 13; MBA genotype D, serovar 9 only; and MBA genotype E comprises serovars 7 and 11. There were no sequence differences between members within each MBA genotype. Further work is required to identify other genes or other regions of the MBA genes that may be used to differentiate U. urealyticum serovars within MBA genotypes A, C and E. A better understanding of the molecular basis of serotype differentiation will help to improve subtyping methods for use in studies of the pathogenesis and epidemiology of these organisms.

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.

In this study, the phylogenetic relationships between the two biovars and 14 serovars of Ureaplasma urealyticum were studied using the sequences of four different genes or genetic regions, namely: 16S rRNA genes; 16S-23S rRNA gene spacer regions; urease gene subunits ureA, ureB, partial ureC and adjoining regions upstream of ureA, ureA-ureB spacer and ureB-ureC spacer; the 5'-ends of the multiple-banded antigen (MBA) genes. U. urealyticum genotypes, based on all four genomic sequences, could be clearly separated into two clusters corresponding with currently recognized biovars 1 and 2. Sequences were generally conserved within each biovar. However, there was heterogeneity within the 5'-end regions of the MBA genes of the four serovars of biovar 1; the sequence of serovar 3 was identical with the previously published sequence and differed by only three bases from that of serovar 14; but there were significant differences between the sequences of serovars 3 and 14 and those of serovars 1 and 6. Based on the phylogenetic analysis, support is given to previous recommendations that the two biovars of U. urealyticum be classified as distinct species, namely U. parvum and U. urealyticum for biovars 1 and 2, respectively. In the future, the relationship between the new species and clinical manifestations of ureaplasma infections should be studied.

Ureaplasma gallorale is a urease-containing mycoplasma (a member of the Mollicutes) which is pathogenic for chickens, from which it was originally isolated. We amplified the 16S rRNA gene of this bacterium and then cloned and sequenced the amplicon. A phylogenetic analysis based on an alignment of the 16S rRNA sequences of U. gallorale and several other Ureaplasma species revealed that U. gallorale is more closely related to Ureaplasma urealyticum than to other Ureaplasma species.

The nucleotide sequences of the spacer regions between the 16S and 23S rRNA genes of six Ureaplasma species were determined following amplification by polymerase chain reaction. The hypothetical secondary structure of the spacer regions was divided into four domains. Domains I and III were highly conserved among different Ureaplasma species but domains II and IV were variable. The intergenic spacer regions of ureaplasmas lacked spacer tRNA genes. Phylogenetic relationships among the Ureaplasma species indicated that the evolutionary history of the Ureaplasma species follows that of the host animal species, suggesting coevolution between ureaplasmas and animals.