Investigation of Infectious Agents Associated with Arthritis by Reverse Transcription PCR of Bacterial Rrna Charles J Cox1, Karen E Kempsell2 and J S Hill Gaston1

Available online http://arthritis-research.com/content/5/1/R1 Research article Open Access Investigation of infectious agents associated with arthritis by reverse transcription PCR of bacterial rRNA Charles J Cox1, Karen E Kempsell2 and J S Hill Gaston1 1Department of Rheumatology, University of Cambridge, Cambridge 2GlaxoSmithKline Medicines Research Centre, Stevenage, UK Corresponding author: J S Hill Gaston (e-mail: [email protected]) Received: 18 July 2002 Revisions received: 11 September 2002 Accepted: 13 September 2002 Published: 11 October 2002 Arthritis Res Ther 2003, 5:R1-R8 (DOI 10.1186/ar602) © 2003 Cox et al., licensee BioMed Central Ltd (Print ISSN 1478-6354; Online ISSN 1478-6362). This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any non-commercial purpose, provided this notice is preserved along with the article's original URL. Abstract In reactive and postinfectious arthritis the joints are generally sequencing the products obtained using universal primers, but sterile but the presence of bacterial antigens and nucleic acids could in some cases be shown to be present by amplifying with has been reported. To investigate whether organisms traffic to species specific primers. This was the case for Yersinia affected joints in these conditions, we performed reverse pseudotuberculosis and Chlamydia trachomatis. However, in transcription PCR using universal primers to amplify any arthritis thought to be related to Campylobacter infection the bacterial 16S rRNA sequences present in synovial fluid. sequences obtained were not from Campylobacter jejuni or Bacterial sequences were detected in most cases, even after C. coli, but from other Campylobacter spp. that are not known treatment of the synovial fluid with DNase, implying the to be associated with reactive arthritis and are probably presence of bacterial RNA and therefore of transcriptionally present as commensals in the gut. We conclude that although active bacteria. Analysis of a large number of sequences rRNA from reactive arthritis associated organisms can be revealed that, as reported in rheumatoid arthritis, most were detected in affected joints, bacterial RNA from many other derived from gut and skin commensals. Organisms known to bacteria is also present, as was previously noted in studies of have triggered arthritis in each case were not found by other forms of inflammatory arthropathy. Keywords: bacterial rRNA, Campylobacter, Chlamydia, reactive arthritis, Yersinia Introduction However, in more recent studies bacterial antigens [5–7] Several forms of inflammatory arthritis are triggered by and, in several cases, bacterial DNA and/or RNA have infection with bacteria, including reactive arthritis (ReA) been identified in synovium or SF from selected patients following certain gastrointestinal or urogenital infections with ReA [8–10] and Lyme arthritis [11]. This implies that [1], Lyme disease [2] and the arthritis associated with organisms associated with these forms of arthritis do streptococcal or neisseria infection. Each of these differs reach the joint, although they may be present there in an from classical septic arthritis in that bacteria cannot readily uncultivable state, and raises the possibility that joint be isolated from the affected joints, although occasionally inflammation is driven by immune responses to bacterial Borrelia burgdorferi has been cultured from the synovial antigens without the need to evoke autoimmune mecha- fluid (SF) of some patients with Lyme arthritis [2,3], and nisms. In Lyme disease both mechanisms have been sug- gonococci or meningococci can be cultured from joints in gested for different phases of disease [12,13]: arthritis the early phase of disease [4]. Because the joint is usually driven by bacterial antigens in early disease that is sterile, these forms of arthritis have been considered to be responsive to antibiotic treatment; and autoimmunity in the result of some form of autoimmune reaction that was antibiotic-resistant, chronic disease caused by cross- triggered by an infection at a site distant from the joint. reactive T cells that are responsive to Borrelia OspA bp = base pairs; RA = rheumatoid arthritis; ReA = reactive arthritis; RT = reverse transcription; SAPHO = synovitis, acne, pustulosis, hyperostosis, osteitis; SF = synovial fluid. R1 Arthritis Research and Therapy Vol 5 No 1 Cox et al. protein and self-antigens such as CD11a. However, the described [14]. To eliminate the risk of contamination with precise role of bacterial antigens in pathogenesis has not bacterial nucleic acids from external sources, all reagents been defined, and it is unclear whether bacterial antigens were prepared using distilled water irradiated with ultra- reach the joint in sufficient quantities to account for the violet at 254 nm for 2 min. Negative controls were major sustained inflammation that can characterize ReA. included at each stage of the RT-PCR procedure in order to ensure that no contamination of samples occurred Using a reverse transcription (RT)-PCR technique to during protocol implementation. detect bacterial 16S rRNA, we recently demonstrated that transcriptionally active bacteria are present in synovial Bacterial ribosomal DNA fragments were amplified from tissue from patients with rheumatoid arthritis (RA) and total cDNA and bacterial genomic DNA by PCR amplifica- other arthropathies [14]. These bacteria were mainly tion using universal, bacterial rRNA specific oligo- derived from commensals that are normally present in the nucleotide primers R1 and R2, as previously described in skin and gut, and we concluded that bacteria engulfed by detail [14]. All PCR negative samples were tested for macrophages can reach the joint when macrophages are potential PCR inhibitors by the addition of 5 ng recruited to the synovial membrane, particularly when Escherichia coli genomic DNA, to their cDNA, and recruitment is increased by joint inflammation. We have retested as described above. If PCR products of the now applied this general technique for the detection of expected size (approximately 400 bp) were detected, then bacteria to SF samples from patients with ReA and other these products were cloned into the PCR product cloning forms of postinfectious arthritis. The data show that the vector pT7-blue (Novagen, Madison, Wisconsin, USA), same commensal bacteria can also be detected in SF according to the manufacturer’s instructions, for sequenc- from these patients, whereas specific disease associated ing and analysis as previously described [14]. bacteria can also be demonstrated in some cases. However, bacterial rRNA sequences from disease associ- Development of specific PCR tests ated bacteria were detected relatively infrequently as com- Having designed specific oligonucleotides that were pared with those from other bacteria also detected in the unique for a given bacteria sequence, these were tested joint. These findings have implications for our understand- against a panel of 2 µl aliquots from 96 sequenced bacte- ing of the pathogenesis of postinfectious arthritis. ria PCR products inserted into recombinant E. coli. Materials and method Using standard reaction mixes and standard cycling times Patients (see above), different primers were tested at annealing SF samples were collected by routine aspiration, mainly temperatures of 55, 60, 63 and 65°C. PCR products were from patients attending the Department of Medicine, then visualized using gel electrophoresis. When the only Addenbrooke’s Hospital, Cambridge, UK. Diagnoses were PCR product detected was from the positive control DNA established by history, serology and culture, as appropri- and the other 95 wells remained negative, a set of primers ate (Table 1). was considered to be specific. If, however, the positive control also failed before specificity was reached, then the Isolation of RNA from synovial fluid and reverse MgCl2 concentration was raised by 0.5 mmol/l increments transcriptase PCR amplification of bacterial rDNA at the first temperature at which the positive control was Total RNA was isolated from 200 µl untreated centrifuged seen to fail. If this failed to get the positive control to work SF using lysis in the presence of 500 µl phenol pH 4.0 before loss of specificity was reached, then the primers and 100 µl chloroform isoamyl alcohol in a Hybaid Ribo- were redesigned and the process repeated. lyserTM (Hybaid, Teddington, Middlesex, UK) according to the manufacturer’s instructions, and recovered by precipi- Results tation with propan-2-ol, dried under appropriate sterile SF samples were collected from 12 patients with various conditions, and then dissolved in diethylene pyrocarbonate- forms of postinfectious arthropathy; 10 had clinical fea- treated water containing 0.1 mmol/l EDTA. Before the RT tures of ReA and two had postmeningococcal or post- step, an aliquot of total RNA was subjected to DNase streptococcal arthritis. An additional two patients were treatment with 1 unit DNase I (Cambridge Bioscience, studied with undifferentiated spondyloarthropathy in which Cambridge, UK), involving incubation at 37°C for 30 min a diagnosis of chronic ReA was considered possible. followed by denaturation of the enzyme by incubation at Patient details are given in Table 1, along with the basis on 75°C for 15 min, whereas the remaining aliquot was not which the diagnosis was made. Total RNA was extracted treated. This was to allow comparisons between DNase from the SF samples and

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