Diverse and related 16S rRNA-encoding DNA sequences in prostate tissues of men with chronic prostatitis

Abstract

Treatment of chronic prostatitis/chronic pelvic pain syndrome is often empirical because clinical culture methods fail to detect prostate-associated pathogens in >90% of patients. Previously, we tested a variety of specific-microorganism PCRs and began a DNA sequence study after we found that 77% of prostatitis patients were PCR positive for prokaryotic rRNA-encoding DNA sequences (rDNAs) despite negative cultures using optimal techniques. In the present study, 36 rDNA clones from 23 rDNA-positive patients were sequenced. This study represents more than twice the total rDNA sequence and more than twice the number of patients in the previous study. The increased number of patients and clones sequenced allowed enhanced phylogenetic analyses and refinements in our view of rDNA species inhabiting the prostate. A continuum of related rDNAs that might be arbitrarily described as two major groups of rDNAs and several minor groups was found. Sequences termed Pros A, identified in 8 (35%) of 23 rDNA-positive patients, grouped with Aeromonas spp. in phylogenetic studies. Sequences termed Pros B, identified in 17 (74%) of 23 rDNA-positive patients, were distinct from previously reported sequences, although all were >90% similar to known gram-negative bacteria. Of the nine patients for whom multiple rDNAs were sequenced, six had biopsy specimens containing rDNAs from more than one species. Four (17%) patients had rDNAs different from those of the Pros A and Pros B groups. Of these four, one patient had rDNA similar to that of Flavobacterium spp., another had rDNA similar to that of Pseudomonas testosteroni, and two patients had rDNAs <70% similar to known rDNAs. These findings suggest that the prostate can harbor bacteria undetectable by traditional approaches. Most of these diverse sequences are not reported in environments outside the prostate. The sequence similarities suggest adaptation of limited groups of bacteria to the microenvironment of the prostate. Further studies may elucidate the relationship of prostate-associated bacteria to chronic prostatitis/chronic pelvic pain syndrome.

FIG. 1

Clustal V multiple sequence alignment and contrast map of six cloned 16S rDNA PCR products from six prostatitis patients. Ahydro, A. hydrophila. Differences are highlighted. Gap penalties were set at 10 (fixed) and 10 (floating). The weighted option of the transition toggle was selected. I5735, previously reported DNA sequence of clone INV5735 (16).

FIG. 2

Venn diagram illustrating the major and minor groups of rDNAs found in prostate biopsy specimens of prostatitis patients. Of the patients whose rDNAs were sequenced, 35% had Pros A type sequences, 74% had Pros B type sequences, and 22% had both Pros A and Pros B sequences (overlap). One patient (4%) had both Pros B and another sequence classified as novel due to lack of similarity with database entries. One patient had rDNA similar to that of Pseudomonas spp., and one patient had rDNA similar to that of Flavobacterium spp., neither of which could be classified as Pros A or Pros B. Two clones from skin at the site of biopsy are not included in the diagram.

FIG. 3

Unrooted dendrogram using the Phylip phylogenetic inference program. Sequences aligned by Clustal V were entered into the Seqboot program of Phylip to generate 1,000 bootstrap replicates consisting of 1,000 sequence alignments each with randomly selected portions of the sequences rendered inactive. The numbers to the left of the nodes indicate the percentage of resulting phylogenetic trees with the nodal topologies shown. These topologies also agreed with the best tree found by using the maximum likelihood program DNAML. H. holobium was entered as the outgroup species, although the phylogeny is unrooted. Horizontal line lengths are proportional to inferred evolutionary distances. Sequences identified by species name are 16S rRNA sequences referenced in GenBank); other entries represent patient prostatic sequences.