Polymerase chain reaction using 16S rRNA gene sequences distinguishes the two biovars of Ureaplasma urealyticum

Abstract

Several fundamental phenotypic and genotypic differences have separated strains of the genital mycoplasma Ureaplasma urealyticum into two clusters or biovars. However, the lack of an easily performed and unambiguous test to discriminate between them has hampered investigation of the relationship between these biovars and disease. We determined the 16S rRNA nucleotide sequence of U. urealyticum 27, the serovar 3 standard and representative of the parvo biovar (serovars 1, 3, 6, and 14). This sequence was compared with the published sequence of U. urealyticum T960, which is the type strain and the serovar 8 standard and is representative of the T960 biovar which is composed of the 10 intervening serovars. Homology between the two sequences was 98.8%; differences were exploited to provide primers for biovar-specific polymerase chain reactions (PCRs). The results of these reactions placed all 14 serovar standard strains into the correct biovar. The PCRs were also applied to 10 cloned and 8 noncloned isolates that had been serotyped earlier. For 16 of them, we deduced their biovars from the serotyping data and then confirmed them by PCR. One unpredictable isolate and one nonserotypeable isolate were also classified as to biovar. Thus, we have developed a method for biotyping U. urealyticum that is applicable to both laboratory-adapted strains and wild-type isolates and that is appropriate for testing large numbers of clinical isolates. The amplification by the T960 biovar PCR protocol of DNAs from ureaplasmas of animals and certain Mycoplasma species suggested that the parvo biovar has diverged from the mainstream of the evolution of this clade.

Biovar Diversity Is Reflected by Variations of Genes Encoding Urease of Ureaplasma urealyticum

“…Consequently, that method classified serovars 10 and 12 into biovar 1. This result conflicts with those of most reports, in which these two serovars are usually classified as biovar 2 on the basis of their susceptibilities to manganese, protein analysis on polyacrylamide gels, Southern blot analysis with rDNA probes, PCR based on 16S rRNA gene sequence and PCR based on MB antigen gene sequence (2,3,6,9,10). Our results with five primer pairs demonstrated that there were variations in urease genes of Ureaplasma urealyticum, and the correlations between the gene variations and biovars was not definite.…”

“…Ureaplasma urealyticum is designated for all Ureaplasma isolated from humans (6). A 14-member serotyping scheme has been widely accepted based on the detection of differences in surface antigenicity (4).…”

“…Biovar 1 is composed of serovars 1, 3, 6 and 14, and the other serovars, 2,4,5,7,8,9,10,11,12 and 13, belong to biovar 2. Because the serovar 8 standard (strain T960) is the type strain of U. urealyticum, Robertson et al designated biovar 2 another name, T960 biovar (6). On the basis of the markedly smaller genomes of biovar 1 strains, parvo biovar was also designated for this biovar.…”

Differentiation of Two Biovars of Ureaplasma urealyticum Based on the 16S-23S rRNA Intergenic Spacer Region

“…Genetic analysis has allowed two biovars to be distinguished on the basis of differences in nucleotide sequences of ureaplasma genes (1,10,15,20,22,26). For example, PCRbased methods have been used to analyze the mba and 16S rRNA genes in U. urealyticum (10,20). In the present study, we examined the 16S-23S rRNA intergenic spacer regions of the 14 serovars of U. urealyticum and showed that this region can also be used for biovar identification.…”

“…Although U. urealyticum strains are most commonly identified by serology, serotyping of ureaplasmas has been hampered because there are no commercially available antisera. Genetic analysis has allowed two biovars to be distinguished on the basis of differences in nucleotide sequences of ureaplasma genes (1,10,15,20,22,26). For example, PCRbased methods have been used to analyze the mba and 16S rRNA genes in U. urealyticum (10,20).…”