Comparative analysis and serovar-specific identification of multiple-banded antigen genes of Ureaplasma urealyticum biovar 1

Abstract

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.

FIG. 1

Algorithm for identification of serovars of U. urealyticum biovar 1 in clinical specimens or cultures. (A) Biotyping of U. urealyticum; (B) serotyping of U. urealyticum biovar 1.

FIG. 2

Multiple sequence alignment of the 5′ ends of MBA gene sequences of U. urealyticum serovars 1, 3, 6, and 14 (ATCC strains). Relevant primers (arrows) and restriction enzyme sites (bars) are shown.

FIG. 3

Results of PCR amplification of MBA genes with primers UMS-125 and UMA269′ followed by restriction enzyme analysis with HinfI. Lanes M, molecular weight markers ΦX174 DNA/HaeIII; lanes 1 and 2, U. urealyticum serovar 1 ATCC strain PCR products before and after digestion by HinfI; lanes 3 and 4, U. urealyticum serovar 1 clinical isolate PCR products before and after digestion by HinfI; lanes 5 and 6, U. urealyticum serovar 1 positive clinical specimen PCR products after digestion by HinfI; lanes 7 and 8, U. urealyticum serovar 6 ATCC strain PCR product before and after digestion by HinfI; lane 9, U. urealyticum serovar 6 clinical isolate PCR product after digestion by HinfI; lanes 10 to 13, PCR products of four clinical specimens digested by HinfI. Lanes 10 and 12 were identified as serovar 1, and lanes 11 and 13 were identified as serovar 6.

FIG. 4

Results of PCR amplification of MBA genes of all 14 serovars of U. urealyticum with primers UMS-125 and UMA269 (specific for serovars 3/14). A positive result is shown by a 442-bp band. Lanes M, molecular weight markers ΦX174 DNA/HaeIII; lanes 1 and 2, U. urealyticum serovars 1 and 2 ATCC strains; lanes 3 and 4, U. urealyticum serovar 3 ATCC strain and clinical isolate; lanes 5 to 14, U. urealyticum serovars 4 to 13 ATCC strains; lanes 15 and 16, U. urealyticum serovar 14 ATCC strain and clinical isolate.

FIG. 5

Results of PCR amplification of MBA genes of all 14 serovars of U. urealyticum with primers UMS-125 and UMA269′ (specific for serovars 1 and 6). A positive result is shown by a 442- or 443-bp band. Lanes M, molecular weight markers ΦX174 DNA/HaeIII; lanes 1 and 2, U. urealyticum serovar 1 ATCC strain and clinical isolate; lanes 3 to 6, U. urealyticum serovars 2 to 5 ATCC strains; lanes 7 and 8, U. urealyticum serovar 6 ATCC strain and clinical isolate; lanes 9 to 16, U. urealyticum serovars 7 to 14 ATCC strains.

FIG. 6

Results of PCR amplification of MBA genes of all 14 serovars of U. urealyticum with primers UMS-54 (specific for serovar 6) and UMA269′. A positive result is shown by a 369-bp band. Lanes M, molecular weight markers ΦX174 DNA/HinfI; lanes 1 and 2, U. urealyticum serovar 1 ATCC strain and clinical isolate; lanes 3 to 6, U. urealyticum serovars 2 to 5 ATCC strains; lanes 7 and 8, U. urealyticum serovar 6 ATCC strain and clinical isolate; lanes 9 to 16, U. urealyticum serovars 7 to 14 ATCC strains.