Reverse Transcriptase-PCR Analysis of Bacterial rRNA for Detection and Characterization of Bacterial Species in Arthritis Synovial Tissue

Abstract

Onset of rheumatoid arthritis (RA) is widely believed to be preceded by exposure to some environmental trigger such as bacterial infectious agents. The influence of bacteria on RA disease onset or pathology has to date been controversial, due to inconsistencies between groups in the report of bacterial species isolated from RA disease tissue. Using a modified technique of reverse transcriptase-PCR amplification, we have detected bacterial rRNA in the synovial tissue of late-stage RA and non-RA arthritis controls. This may be suggestive of the presence of live bacteria. Sequencing of cloned complementary rDNA (crDNA) products revealed a number of bacterial sequences in joint tissue from each patient, and from these analyses a comprehensive profile of the organisms present was compiled. This revealed a number of different organisms in each patient, some of which are common to both RA and non-RA controls and are probably opportunistic colonizers of previously diseased tissue and others which are unique species. These latter organisms may be candidates for a specific role in disease pathology and require further investigation to exclude them as causative agents in the complex bacterial millieu. In addition, many of the detected bacterial species have not been identified previously from synovial tissue or fluid from arthritis patients. These may not be easily cultivable, since they were not revealed in previous studies using conventional in vitro bacterial culture methods. In situ hybridization analyses have revealed the joint-associated bacterial rRNA to be both intra- and extracellular. The role of viable bacteria or their nucleic acids as triggers in disease onset or pathology in either RA or non-RA arthritis controls is unclear and requires further investigation.

Molecular Identification of Microorganisms from Endodontic Infections

“…Detection of viable bacteria would have to be determined using cDNA as a template for PCR and would therefore require a reverse transcription-PCR method. Such techniques are common to studies on environmental isolates but have also been used recently on a large scale to detect potential uncultivable bacteria in infected synovial tissue from patients with arthritis (20).…”

Detection ofMycobacterium tuberculosisGroup Organisms in Human and Mouse Joint Tissue by Reverse Transcriptase PCR: Prevalence in Diseased Synovial Tissue Suggests Lack of Specific Association with Rheumatoid Arthritis

“…Healthy controls were not age and sex matched to the RA patient group; however, the trauma specimens were unlikely to have features of joint disease pathology in common with arthritis patients of many years duration. Full microbiological analyses of patients from cohort 1 have been published in detail elsewhere (28). Synovial tissue was obtained in cohort 2 patients with from late-stage RA and OA, plus patient controls with arthritis due to other causes (Table 4), either at surgery for joint replacement, from power tool washings for debridement of inflamed synovium, or by arthroscopic biopsy.…”

“…The presence of multiple bacterial species in the tissues of patients with RA, but also patients with other arthropathies, has called into question the involvement of these organisms in the underlying pathological processes and has implied general colonization as a consequence of the compromise of chronically diseased and inflamed tissue (28). Many of the bacterial species detected were commensal organisms with low pathogenic potential, which presumably had trafficked from other body sites.…”

“…Work from our laboratory has shown the presence of possibly live bacteria in the joints of patients with a variety of different arthropathies, using reverse transcriptase PCR (RT-PCR) for amplification of bacterial rRNAs (28). Detailed sequencing analyses of the amplified products has shown the picture to be considerably more complex that was once thought, with the detection of multiple bacterial species in each patient.…”

Molecular detection of transcriptionally active bacteria from failed prosthetic hip joints removed during revision arthroplasty

“…In order to achieve this, bacterial mRNA was detected within the biofilm of the failed prosthetic hip joint, rather than DNA as in our first study. This approach has been used previously to identify transcriptionally active bacteria in the synovial tissue of patients with rheumatoid arthritis and other arthropathies [15,16]. Conventional microbiological culture was also carried out in order to identify as many bacterial species as possible that are associated with prosthetic hip joint infections.…”