Phylogeny-based rapid identification of mycoplasmas and ureaplasmas from urethritis patients

Abstract

Some strains of mycoplasmas and ureaplasmas (family Mycoplasmataceae) are associated with nongonococcal urethritis (NGU) or other genitourinary infections. We have developed a rapid and reliable method of identifying the presence and prevalence of mycoplasmas and ureaplasmas in men with NGU. This method is based on the amplification of a part of the 16S rRNA gene by PCR and phylogenetic analysis. A portion of the 16S rRNA gene from 15 prototype strains was amplified with a set of common primers, and their nucleotides were sequenced. The nucleotide sequence of the V4 and V5 regions was analyzed by the neighbor-joining method. The 15 prototype strains were grouped into three distinct clusters, allowing us to clearly segregate the strains into distinct lineages. To determine the prevalence of these pathogens among patients with NGU, this protocol was tested with 148 urine samples. Amplifications were observed for 42 samples, and their nucleotide sequences were analyzed along with those of the 15 prototype strains. The phylogenetic tree thus constructed indicated that 15 of the 42 formed a cluster with Mycoplasma genitalium. Among the remaining specimens, 2 formed a cluster with Mycoplasma hominis, 19 with Ureaplasma urealyticum, and 5 with Ureaplasma parvum; the remaining sample contained both M. genitalium and U. urealyticum. This phylogeny-based identification of mycoplasmas and ureaplasmas provides not only a powerful tool for rapid diagnosis but also the basis for etiological studies of these pathogens.

FIG. 1.

Phylogenetic analysis of prototype strains of human mycoplasmas and ureaplasmas. The nucleotide sequences including the V4 to V5 regions (approximately 250 bp) from the 15 prototype strains were analyzed by the neighbor-joining method. The numbers at the nodes represent the percentage of 1,000 bootstrap pseudoreplicates that contained the cluster distal to the node.

FIG. 2.

Phylogenetic analyses of 14 serovars of ureaplasmas. The nucleotide sequences including the V4 to V5 regions of the isolates were determined and then analyzed by the neighbor-joining method. The numbers at the nodes represent the percentage of 1,000 bootstrap pseudoreplicates that contained the cluster distal to the node. Fourteen serovars of ureaplasmas formed two distinct clusters, U. parvum (previously U. urealyticum biovar 1) and U. urealyticum (previously U. urealyticum biovar 2).

FIG. 3.

Phylogenetic analyses of mycoplasmas and ureaplasmas from urine specimens. Eight representative nucleotide sequences of 57 DNA fragments from urine specimens were analyzed with the 15 prototype strains as for Fig. 1. The numbers at the nodes represent the percentages of 1,000 bootstrap pseudoreplicates that contained the cluster distal to the node. The numbers in the parentheses indicate the numbers of PCR fragments that showed nucleotide sequences identical to those from urine samples.