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Open Access Research article

Broad-range PCR, cloning and sequencing of the full 16S rRNA gene for detection of bacterial DNA in synovial fluid samples of Tunisian patients with reactive and undifferentiated arthritis

Mariam Siala1, Radhouane Gdoura1, Hela Fourati2, Markus Rihl3, Benoit Jaulhac4, Mohamed Younes5, Jean Sibilia4, Sofien Baklouti2, Naceur Bargaoui5, Slaheddine Sellami6, Abdelghani Sghir78 and Adnane Hammami1*

Author Affiliations

1 Laboratoire de Recherche 'Micro-organismes et Pathologie Humaine', EPS Habib Bourguiba, Rue El Ferdaous, 3029 Sfax, Tunisie

2 Service de Rhumatologie Hôpital Hedi Chaker, Avenue Majida Boulila, 3029 Sfax, Tunisie

3 Hannover Medical School (MHH), Clinic for Immunology and Rheumatology, 30625 Hannover; Germany

4 Laboratoire de Physiopathologie des Interactions Hôte-bactérie, UPRES-EA 3432, Faculté de Médecine, Université Louis-Pasteur, rue Koeberlé, 67000 Strasbourg, France

5 Service de Rhumatologie, EPS Fattouma Bourguiba, Rue 1er Juin, 5019 Monastir, Tunisie

6 Service de Rhumatologie, EPS La Rabta, rue 7051 Centre Urbain Nord, 1082 Tunis, Tunisie

7 CNRS-UMR 8030, CEA-Genoscope, rue Gaston Crémieux, 91000 Évry, France

8 University of Evry Val d'Essonne, Boulevard François Mitterrand, 91025 Évry Cedex, 91000 Évry, France

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Arthritis Research & Therapy 2009, 11:R102  doi:10.1186/ar2748

Published: 1 July 2009

Abstract

Introduction

Broad-range rDNA PCR provides an alternative, cultivation-independent approach for identifying bacterial DNA in reactive and other form of arthritis. The aim of this study was to use broad-range rDNA PCR targeting the 16S rRNA gene in patients with reactive and other forms of arthritis and to screen for the presence of DNA from any given bacterial species in synovial fluid (SF) samples.

Methods

We examined the SF samples from a total of 27 patients consisting of patients with reactive arthritis (ReA) (n = 5), undifferentiated arthritis (UA) (n = 9), rheumatoid arthritis (n = 7), and osteoarthritis (n = 6) of which the latter two were used as controls. Using broad-range bacterial PCR amplifying a 1400 bp fragment from the 16S rRNA gene, we identified and sequenced at least 24 clones from each SF sample. To identify the corresponding bacteria, DNA sequences were compared to the EMBL (European Molecular Biology Laboratory) database.

Results

Bacterial DNA was identified in 20 of the 27 SF samples (74, 10%). Analysis of a large number of sequences revealed the presence of DNA from more than one single bacterial species in the SF of all patients studied. The nearly complete sequences of the 1400 bp were obtained for most of the detected species. DNA of bacterial species including Shigella species, Escherichia species, and other coli-form bacteria as well as opportunistic pathogens such as Stenotrophomonas maltophilia and Achromobacter xylosoxidans were shared in all arthritis patients. Among pathogens described to trigger ReA, DNA from Shigella sonnei was found in ReA and UA patients. We also detected DNA from rarely occurring human pathogens such as Aranicola species and Pantoea ananatis. We also found DNA from bacteria so far not described in human infections such as Bacillus niacini, Paenibacillus humicus, Diaphorobacter species and uncultured bacterium genera incertae sedis OP10.

Conclusions

Broad-range PCR followed by cloning and sequencing the entire 16S rDNA, allowed the identification of the bacterial DNA environment in the SF samples of arthritic patients. We found a wide spectrum of bacteria including those known to be involved in ReA and others not previously associated with arthritis.