Search

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.

Ureaplasma urealyticum is a causative agent of nongonococcal urethritis and is implicated in the pathogenesis of several other diseases. The species is divided into 14 serovars and two biovars, of which biovar 1 is most commonly isolated from clinical specimens. Reported associations between individual serovars and diseases have been difficult to confirm because of practical difficulties with serotyping. The multiple-banded antigen (MBA) is the predominant U. urealyticum antigen recognized during infections in humans and probably has a significant role in virulence. The 5' end of the MBA gene is relatively conserved but contains biovar, and possibly serovar, specificity. The 5' ends of the MBA genes of standard strains of U. urealyticum biovar 1, consisting of serovars 1, 3, 6, and 14, were amplified, cloned into pUC19, and sequenced to identify serovar-specific differences. The 5' end of the MBA gene sequence of serovar 3 was identical with the previously published sequence and differed by only three bases from that of serovar 14. Significant differences between the MBA gene sequences allowed biovar 1 to be divided into two subgroups, containing serovars 3/14 and serovars 1 and 6, respectively, using primers UMS-125-UMA269 and UMS-125-UMA269'. Serovars 1 and 6 were distinguished by restriction enzyme analysis of the amplicon and/or by PCR specific for serovar 6. These methods were used to identify and type U. urealyticum in 185 (46.3%) of 400 genital specimens from women. Biovar 1 was detected in 89.2% and biovar 2 in 18.3% of positive specimens. Of 165 specimens containing U. urealyticum biovar 1, 22.2% contained more than one serovar and 46.7, 46.1, and 25.5% contained serovars 1, 3/14, and 6, respectively. U. urealyticum was found in a significantly higher proportion of pregnant women than in sex workers and other women attending a sexually transmissible diseases clinic (P < 0.01). The methods described are relatively rapid, practicable, and specific for serotyping isolates and for direct detection and identification of individual serovars in clinical specimens containing U. urealyticum biovar 1.

Sequencing of mba gene fragments of reference strains of Ureaplasma urealyticum serovars 1, 3, 6, 14 and 8, in addition to 33 clinical U. urealyticum isolates is reported. A phylogenetic tree deduced from alignment of these sequences clearly demonstrates two major clusters (confidence limit 100%), which equate to the parvo and T960 biovars, and five types, which have been designated mba 1, 3, 6, 8 and X. These relationships are supported by bootstrap analysis. Polymorphisms within the mba fragment of types mba 1, 3 and 6 were used to define nine subtypes (mba 1a, 1b, 3a, 3b, 3c, 3d, 3e, 6a and 6b), thus facilitating high resolution typing of U. urealyticum. Inclusion of reference strains for serovars 1, 3, 6 and 8 in the mba typing scheme showed that the results of this analysis are broadly consistent with currently accepted serotyping. In addition, a ure gene fragment from nine of the clinical isolates was amplified and sequenced. Comparisons of the sequences clearly distinguished the two biovars of U. urealyticum; however, this fragment was invariant within the parvo biovar. This study has shown that the mba sequence can reveal the fine details of the relationships between U. urealyticum isolates and also supports the significant evolutionary gap between the two biovars.

Ureaplasma urealyticum is a common commensal of the female lower urogenital tract, yet it has been shown to be an important cause of chorioamnion infection, respiratory and central nervous system disease, and death in premature infants. It has been suggested that only certain serovars are capable of producing invasive disease. However, we previously showed that many serotypes are invasive and that perhaps antigen variability and host factors are more important determinants of ureaplasma infections than are different serotypes per se. The molecular characterization in this report describes a mechanism available to ureaplasmas for producing antigen variation. That antigen, designated MB and previously identified on U. urealyticum, contains serovar-specific and cross-reactive epitopes, is produced both in vitro and in vivo, is a predominant antigen recognized during ureaplasma infections of humans, undergoes a high rate of size variation in vitro, and is size variable on invasive ureaplasma isolates. In the present study, we cloned and sequenced the gene of the MB antigen from serovar 3, the serovar most commonly isolated from humans. The 3' two-thirds of the gene was shown to contain identical 18-nucleotide tandem repeats. PCR analysis and direct sequencing of two variants indicated that alterations within this repeat region are responsible for the size variation of the MB antigen. Intact recombinant serovar 3 MB antigen and truncated products, expressed by coupled in vitro transcription and translation of the cloned gene, were immunoprecipitated by both a serovar-specific monoclonal antibody and the serum of a U. urealyticum-infected patient, and these results identified the repeat region of the MB antigen as serovar defining. Resolution of the precise amino acids responsible for specific epitopes and characterization of similar genes in the other serovars should yield reagents useful in elucidating the role of antigen size variants in disease production and the role of specific antibody in protection from ureaplasma disease.

Ureaplasma urealyticum is a commensal organism of the lower genital tract of females, but in a subpopulation of individuals, it can invade the upper genital tract. It is a significant cause of chorioamnionitis and neonatal morbidity and mortality. There are 14 recognized serovars of U. urealyticum; these can be divided into two distinct clusters or biovars. Biovar 1 is composed of serovars 1, 3, 6, and 14, Biovar 2 is composed of serovars 2, 4, 5, 7, 8, 9, 10, 11, 12, and 13. We previously identified a surface antigen, the multiple-banded (MB) antigen, which contains both serovar-specific and cross-reactive epitopes. Genotypic characterization of the C-terminal region of the MB antigen of serovar 3 indicates that serovar specificity and MB antigen size variation reside in that domain. In the present study, we used PCR analysis with primers derived from the serovar 3 MB antigen gene DNA sequence to determine if the MB antigen gene was present in the remaining 13 reference serovars as well as in invasive clinical isolates. The results indicated that not only was the MB antigen gene present in all serovars but that the genes' 5' regions were markers of biovar specificity and diversity. Further analysis of this region should reveal the phylogenetic relationship among serovars of U. urealyticum and, possibly, their invasive potential.