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.

FIG. 1

Results of RT-PCR of bacterial 16S rRNA from patient RNA samples, amplification products visualized by agarose gel electrophoresis, and ethidium bromide staining.

FIG. 2

Diagrammatic representation of the bacterial species identified in RA patients by sequencing of cloned 16S crDNA amplicons. Each genus is represented by colour coding, and species are depicted by initials derived from abbreviated species names (Table 4). Section sizes are representative of the total number of sequences for that species in each patient.

FIG. 3

Diagrammatic representation of the bacterial species identified in the OA and UA patients. The outline for genus and species representation is given in Fig. 2.

FIG. 4

Diagrammatic representation of the total numbers of bacterial species unique to each patient group. (A) RA patients; (B) non-RA patients. Dark-shaded segments indicated species unique to that disease and found in more than one patient. Lighter-shaded segments indicate species unique to that disease and found in one patient only. Blank segments indicate species common to both patient groups.

FIG. 5

Cryostat tissue sections from patient 8, stained with bacterial Gram stain. (A) Extracellular microcolony of staphylococci. Magnification, ×100. (B) cell-associated bacteria staining unconventionally brown by this staining technique. Magnification, ×40.

FIG. 6

Control and undifferentiated arthritis cryostat tissue sections stained with bacterium-specific stains or by in situ hybridization using digoxgenin-labeled bacterial 16S rRNA oligonucleotides ISH1 to ISH3. (A) Control mouse liver stained by in situ hybridization. (B) Control human kidney stained by in situ hybridization. (C) M. tuberculosis-infected mouse lung stained with mycobacterium-specific Ziehl-Nielson Carbolfuschein. (D) M. tuberculosis-infected mouse was stained by in situ hybridization. (E) Section from negative undifferentiated arthritis patient 16 stained by in situ hybridization. (F) Section from undifferentiated arthritis patient 15 stained by in situ hybridization. Note the intracellular staining (IC) within a focus of inflammatory cells. Magnifications, ×20 (A), ×40 (B and F), and ×10 (C to E).

FIG. 7

RA and OA arthritis cryostat tissue sections stained by in situ hybridization using digoxigenin-labeled bacterial 16S rRNA oligonucleotides ISH1 to ISH3. (A) RA patient 7. Note the heavy staining within a focus of what appear to be inflammatory cells. (B) RA patient 8. Note the sparse staining in isolated cells. (C) Patient 12 sample a. (D) Patient 12 sample b. (E) Patient 12 sample g. (F) Patient 12 sample e. Signals correlate with those obtained by RT-PCR. Magnifications, ×20.