Comparative Analysis and Serovar-Specific Identification of Multiple-Banded Antigen Genes of Ureaplasma urealyticum Biovar 1

Bacterial strains and plasmids.The reference strains used were U. urealyticum serovar 1 (ATCC 27813), serovar 2 (ATCC 27814), serovar 3 (ATCC 27815), serovar 4 (ATCC 27816), serovar 5 (ATCC 27817), serovar 6 (ATCC 27818), serovar 7 (ATCC 27819), serovar 8 (ATCC 27618), serovar 9 (ATCC 33175), serovar 10 (ATCC 33699), serovar 11 (ATCC 33695), serovar 12 (ATCC 33696), serovar 13 (ATCC 33698), and serovar 14 (ATCC 33697). Clinical isolates of serovars 1 to 14, which had been serotyped by conventional methods, were kindly provided by H. L. Watson (Department of Microbiology, University of Alabama at Birmingham, Birmingham) Escherichia coli DH5α was the host for plasmid pUC19, which was obtained from the Institute of Genetics, Chinese Academy of Science.

Clinical specimens.Cervical swabs from 200 female sex workers and 100 other female clients attending the Sexually Transmissible Diseases (STD) Clinic and 100 pregnant women attending the Antenatal Clinic of the First Hospital of Beijing Medical University were collected to test the practical usefulness of the direct detection, biotyping, and serotyping methods. DNA was prepared for PCR (see below) as soon as possible after receipt of specimens in the laboratory. Cultures were not performed for these specimens.

Oligonucleotide primers.Oligonucleotide primers UMS-125, UMA226, UMS51, and UMA427, based on the previously published sequence of the serovar 3 MBA gene (19, 22), were used for amplification of the 5′ ends of the U. urealyticum serovar 1, 3, 6, and 14 MBA genes. UMS-125 (GTA TTT GCA ATC TTT ATA TGT TTT CG) and UMA226 (CAG CTG ATG TAA GTG CAG CAT TAA ATT C) were used without modification. UMS51 and UMA427 were modified as follows. (i) Primers UMS51 and UMA427 were modified by adding BamHI andEcoRI restriction enzyme sites, respectively (underlined in the sequences below), to facilitate cloning of the coding DNA sequences of the 5′ ends of the serovar 1, 3, and 14 MBA genes. (ii) In ureaplasmas, the sequence TGA encodes tryptophan (amino acid 13), but in E. coli it represents a stop codon. Therefore, a point mutation was introduced into UMS51 (at base 38) to change the sequence to TGG, which also encodes tryptophan. The modified primer sequences used were UMS51, TGT GGA TCC TTC TGG GCT ATG ACA TTA GGT GTT ACC (BamHI site underlined) and UMA427, CTCGAA TTC ACC TGG TTG TGT AGT TTC AAA GTT CAC (EcoRI site underlined).

The additional primers UMA269 (serovar 3/14 specific), UMA269′ (serovar 1 and 6 specific), and UMS-54 (serovar 6 specific) were designed, using the sequence data generated by this study, and were used for PCR identification (see below). The sequences of these primers are as follows: UMA269 (serovar 3/14), CTA AAT GAC CTT TTT CAA GTG TAC; UMA269′ (serovars 1 and 6), CCA AAT GAC CTT TTG TAA CTA GAT; and UMS-54 (serovar 6), CTT AGT GTT CAT ATT TTT TAC TAG.

DNA preparations.Cells from 0.5 ml of ureaplasma broth (10B) cultures of each U. urealyticum serovar were harvested in late logarithmic growth phase by centrifugation at 14,000 × g for 20 min; clinical specimens were processed directly. DNA was isolated from both cultures and clinical specimens by treatment with 500 μl of digestion buffer (10 mM Tris-HCl, pH 8.0, 0.45% Triton X-100, and 0.45% Tween 20) and proteinase K (100 μg/ml) at 55°C for 1 h and then extraction with phenol-chloroform-isoamyl alcohol (25:24:1) and chloroform-isoamyl alcohol (24:1). DNA was precipitated with 1/10 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol. The washed and dried pellets were hydrated in 200 μl of ultrapure sterile water.

PCR.The 25-μl amplification reaction mixtures contained 2.5 μl of 10× PCR buffer (1× PCR buffer is 10 mM Tris-HCl [pH 8.8 at 25°C], 1.5 mM MgCl₂, 50 mM KCl, and 0.1% Triton X-100), 0.5 U of Taq polymerase (Finnzymes OY, Espoo, Finland), 200 μM (each) deoxynucleoside triphosphate (dATP, dCTP, dGTP, and dTTP) (Boehringer, Mannheim, Germany), 10 pmol of each primer, 5 μl of sample DNA, and ultrapure sterile water added to 25 μl. In each reaction, positive and negative controls were processed in parallel with the tested samples to detect possible inhibition or contamination.

The denaturation, annealing, and elongation temperatures and times used were 95°C for 30 s, 58°C for 30 s, and 72°C for 1 min, respectively, for 40 cycles with a Perkin-Elmer thermocycler. Eight microliters of PCR product was analyzed by electrophoresis on 2.0% agarose gels which were stained with 0.5 μg of ethidium bromide/ml. A visible band of the appropriate size on UV translumination was considered a positive result.

Cloning and sequencing.The amplified fragments of the 5′ ends of U. urealyticum MBA genes (amplified with UMS-125–UMA226 for serovar 6 and UMS51-UMA427 for serovars 1, 3, and 14) were ligated to plasmid vector pUC19, which had been digested bySmaI. The ligation products were transformed into E. coli DH5α, and positive transformants were identified by PCR, using the primers UMS51-UMA427 for serovars 1, 3, and 14 and UMS-125–UMA226 for serovar 6. The positive clones were sequenced in two directions with the ABI 373A DNA-sequencing system.

To confirm the results of cloning experiments, the PCR products of both sets of primers for ATCC reference strains and clinical isolates of all four serovars, 1, 3, 6, and 14, were directly sequenced in two directions with the ABI 373A DNA-sequencing system.

Identification of serovars by PCR with restriction enzyme analysis and serovar-specific PCR.Primers UMS-125, UMA226, UMA269, and UMA269′ were used to amplify DNA from reference (ATCC) strains, from clinical isolates that had been serotyped previously by conventional methods, and directly from clinical specimens. Primers UMS-125 and UMA226 were used to detect and biotype U. urealyticum. Primers UMS-125 and UMA269 were specific for serovars 3/14; primers UMS-125 and UMA269′ were specific for serovars 1 and 6. Two different methods were used to further identify U. urealyticum serovars 1 and 6 (Fig.1).

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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.

(i) Restriction enzyme analysis.Ten microliters of the PCR product of primers UMS-125 and UMA269′ was treated with the restriction enzyme HinfI (New England Biolabs, Beverly, Mass.) and analyzed by electrophoresis. Those that were cut by the enzyme were identified as serovar 1, those not cut were identified as serovar 6, and those that were partially digested were assumed to be mixed serovars 1 and 6.

(ii) Confirmatory PCR for serovar 6.Primers UMS-54 (specific for serovar 6) and UMA269′ were used in a separate PCR to confirm the identity of serovar 6-positive isolates or specimens.

Nucleotide sequence accession numbers.The sequence data obtained have been accepted by the GenBank nucleotide sequence database and have been assigned accession no. AF056982 (serovar 14), AF056983(serovar 1), and AF056984 (serovar 6). They will also appear in EMBL in Europe and the DNA database Bank of Japan.