Bacterial DNA signatures in carotid atherosclerosis represent both commensals and pathogens of skin origin.

Infectious agents have been suggested to be involved in atherosclerosis. By using a novel subtraction broad-range PCR approach, we defined bacterial DNA signatures in surgically removed sterile carotid artery endarterectomy plaques of patients with carotid atherosclerosis. Eighty partial bacterial 16S rDNA nucleotide sequences from eight patients were studied. Furthermore, 34 clones representing 21 bacterial sequence-types from the reagents used for DNA extraction and PCR amplification were determined. After subtraction of these potential methodological contaminants, 23 bacterial sequence-types were considered as clinically relevant findings. The most prominent phylum, Actinobacteria, accounted for 74% of these relevant sequences. Furthermore, according to the Human Microbiome project database, interestingly, nearly all (94%) of the sequences were associated with the human skin microbiome.

Bacterial signatures in atherosclerotic lesions represent human commensals and pathogens.

BACKGROUND: Various studies have suggested the involvement of infectious agents in chronic inflammatory diseases, including atherosclerosis. By using a novel subtraction broad-range PCR approach, we defined bacterial DNA signatures in surgically removed sterile abdominal aorta samples of patients with aortic atherosclerosis. METHODS: Partial bacterial 16S rDNA nucleotide sequences were determined using broad-range PCR from aortic samples of 20 patients, and from appropriate methodological controls. In all, 160 sequences from 16 clone libraries were studied. RESULTS: After subtraction analysis 16 clinically relevant bacterial sequence-types were identified among the patient samples, whereas 29 were discarded as potential methodological contaminants. On average 2.2+/-1.2 different bacterial sequence-types were present in the nine true PCR-positive atheroma samples. CONCLUSIONS: Many studies have reported the presence of a variety of bacterial sequences from atherosclerotic lesions. However, the results obtained with these PCR technologies may have been skewed by methodological contaminants. After our subtraction approach, 63% of the remaining sequence-types from sites of aortic atherosclerosis were related to those of known human pathogens. This may imply that advanced atherosclerotic plaques accumulate bacterial DNA.

Methanogenic Archaea and human periodontal disease.

Archaea have been isolated from the human colon, vagina, and oral cavity, but have not been established as causes of human disease. In this study, we reveal a relationship between the severity of periodontal disease and the relative abundance of archaeal small subunit ribosomal RNA genes (SSU rDNA) in the subgingival crevice by using quantitative PCR. Furthermore, the relative abundance of archaeal small subunit rDNA decreased at treated sites in association with clinical improvement. Archaea were harbored by 36% of periodontitis patients and were restricted to subgingival sites with periodontal disease. The presence of archaeal cells at these sites was confirmed by fluorescent in situ hybridization. The archaeal community at diseased sites was dominated by a Methanobrevibacter oralis-like phylotype and a distinct Methanobrevibacter subpopulation related to archaea that inhabit the gut of numerous animals. We hypothesize that methanogens participate in syntrophic relationships in the subgingival crevice that promote colonization by secondary fermenters during periodontitis. Because they are potential alternative syntrophic partners, our finding of larger Treponema populations sites without archaea provides further support for this hypothesis.

Analysis of conserved non-rRNA genes of Tropheryma whipplei.

The causative agent of Whipple's disease, Tropheryma whipplei, is a slow-growing bacterium that remains poorly-understood. Genetic characterization of this organism has relied heavily upon rRNA sequence analysis. Pending completion of a complete genome sequencing effort, we have characterized several conserved non-rRNA genes from T. whipplei directly from infected tissue using broad-range PCR and a genome-walking strategy. Our goals were to evaluate its phylogenetic relationships, and to find ways to expand the strain typing scheme, based on rDNA sequence comparisons. The genes coding for the ATP synthase beta subunit (atpD), elongation factor Tu (tuf), heat shock protein GroEL (groEL), beta subunit of DNA-dependent RNA polymerase (rpoB), and RNase P RNA (rnpB) were analyzed, as well as the regions upstream and downstream of the rRNA operon. Phylogenetic analyses with all non-rRNA marker molecules consistently placed T. whipplei within the class, Actinobacteria. The arrangement of genes in the atpD and rpoB chromosomal regions was also consistent with other actinomycete genomes. Tandem sequence repeats were found upstream and downstream of the rRNA operon, and downstream of the groEL gene. These chromosomal sites and the 16S-23S rRNA intergenic spacer regions were examined in the specimens of 11 patients, and a unique combination of tandem repeat numbers and spacer polymorphisms was found in each patient. These data provide the basis for a more discriminatory typing method for T. whipplei.

Prevalence of bacteria of division TM7 in human subgingival plaque and their association with disease.

Members of the uncultivated bacterial division TM7 have been detected in the human mouth, but little information is available regarding their prevalence and diversity at this site. Human subgingival plaque samples from healthy sites and sites exhibiting various stages of periodontal disease were analyzed for the presence of TM7 bacteria. TM7 ribosomal DNA (rDNA) was found in 96% of the samples, and it accounted for approximately 0.3%, on average, of all bacterial rDNA in the samples as determined by real-time quantitative PCR. Two new phylotypes of this division were identified, and members of the division were found to exhibit filamentous morphology by fluorescence in situ hybridization. The abundance of TM7 rDNA relative to total bacterial rDNA was higher in sites with mild periodontitis (0.54% +/- 0.1%) than in either healthy sites (0.21% +/- 0.05%, P < 0.01) or sites with severe periodontitis (0.29% +/- 0.06%, P < 0.05). One division subgroup, the I025 phylotype, was detected in 1 of 18 healthy samples and 38 of 58 disease samples. These data suggest that this phylotype, and the TM7 bacterial division in general, may play a role in the multifactorial process leading to periodontitis.

Broad-range bacterial detection and the analysis of unexplained death and critical illness.

Broad-range rDNA polymerase chain reaction (PCR) provides an alternative, cultivation-independent approach for identifying pathogens. In 1995, the Centers for Disease Control and Prevention initiated population-based surveillance for unexplained life-threatening infections (Unexplained Death and Critical Illness Project [UNEX]). To address the causes of UNEX cases, we examined 59 specimens from 46 cases by using broad-range bacterial 16S rDNA PCR and phylogenetic analysis of amplified sequences. Specimens from eight cases yielded sequences from Neisseria meningitidis (cerebrospinal fluid from two patients with meningitis), Streptococcus pneumoniae (cerebrospinal fluid from one patient with meningitis2 and pleural fluid from two patients with pneumonia), or Stenotrophomonas maltophilia (bone marrow aspirate from one patient with pneumonia). Streptococcus pneumoniae rDNA sequence microheterogeneity was found in one pleural fluid specimen, suggesting the presence of multiple strains. In conclusion, known bacterial pathogens cause some critical illnesses and deaths that fail to be explained with traditional diagnostic methods.