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

ar2748 ARJ 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 Siala Mariam mariamsiela@voila.fr Gdoura Radhouane gdourar@yahoo.com Fourati Hela hela.fourati@rns.tn Rihl Markus Rihl.Markus@mh-hannover.de Jaulhac Benoit benoit.jaulhac@medecine.u-strasbg.fr Younes Mohamed mohamed_youn@yahoo.fr Sibilia Jean jean.sibilia@wanadoo.fr Baklouti Sofien sofien.baklouti@rns.tn Bargaoui Naceur naceur.bergaoui@rns.tn Sellami Slaheddine slaheddine.sellami@rns.tn Sghir Abdelghani sghir@genoscope.cns.fr Hammami Adnane adnene.hammami@rns.tn

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

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

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

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

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

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

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

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

Arthritis Research & Therapy 1478-6354 2009 11 4 R102 http://arthritis-research.com/content/11/4/R102 19570210 10.1186/ar2748 08 4 2009 21 5 2009 29 5 2009 01 7 2009 01 7 2009 2009 Siala et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Introduction

The actual pathogenic event initiating arthritis is largely unknown. For several forms of arthritis, an infectious etiology has been postulated 123. In particular, reactive arthritis (ReA) is known to be triggered by a variety of bacteria. For Salmonella, Yersinia, and Chlamydia, a persistent infection has been hypothesized due to the intraarticular presence of bacterial antigens, DNA, and/or RNA 456. There is also evidence that undifferentiated arthritis is a form of ReA ('forme fruste') possibly due to a preceding but asymptomatic infection 78. PCR using universal 16S rRNA primers is a highly sensitive tool allowing detection of unknown, that is unsuspected, pathogens relating to all eubacterial species 91011. This tool has been used before in patients with ReA, undifferentiated arthritis (UA), and other arthropathies. However, in most studies, the PCR products were of sufficient length to determine the genus of the bacteria in the synovial samples, but were not long enough to identify the species level 121314.

We previously used the broad-range PCR, cloning and sequencing the entire 16S rDNA and demonstrated the presence of a large number of bacterial DNA in the synovial tissue (ST) of patients with ReA, UA, and other arthropathies 15. These bacterial DNA were mainly derived from commensals that are normally present in the skin and gut. We also detected DNA of specific bacterial groups that have not been detected in arthritis samples or in human infections so far, suggesting that these new bacteria possibly could have a pathogenic relevance, particularly with regard to the ST. The detection of such a variety of bacterial groups after cloning and near full-length 16S rDNA sequencing obtained in ST samples has raised the question if the identical bacterial DNA communities could reside in both the ST and the synovial fluid (SF) of matched arthritis patients 15. In addition, the composition of bacterial DNA from ST samples has not been compared comprehensively with that of matched SF samples in arthritis patients. Besides, a detailed analysis of SF bacterial DNA and their comparison with those from corresponding ST samples might help to determine whether intraarticular bacterial DNA might change over time between the two synovial compartments.

As opposed to SF 1216, the bacterial DNA communities in the ST are well documented 1314171819 and to our knowledge, there is no study that has amplified the entire 16S rDNA from SF samples.

Accordingly, we now continue our study using PCR as well as cloning and sequencing of the entire 16S rDNA to identify any bacterial DNA potentially present in the SF samples of patients with ReA, UA, and other forms of arthritis who were also analyzed previously 15.

Materials and methods

Patients and synovial fluid samples

Twenty-seven patients with active arthritis and knee effusion gave informed consent and were included in the study, which was approved by the local ethical committees. ST samples of these patients were used in a previous study 15. The clinical characteristics as well as technical aspects regarding prevention of contamination during sampling have recently been published in detail 15.

Among the 27 patients included in the study, five were diagnosed with ReA, and nine with undifferentiated arthritis (UA); we also included seven patients with rheumatoid arthritis (RA) and six with osteoarthritis (OA) who served as controls. In the current study, the 27 patients were identical to the 27 patients analyzed in our previously published study (from patient 2 to patient 28) 15. The clinical features and demographic characteristics of the patients are summarized in Table 1. SF samples were aspirated by standard needle puncture, snap frozen in liquid nitrogen, and stored at -80°C until analysis.

Table 1

Demographic and clinical features of the study patients

Diagnosis (patients; n = 27)

Median disease duration, months (range)

Actual age or median age, years (range)

Sex ratio (M/F)

Clinical details

ReA (n = 5)

2.4 (1 to 6)

32.4 (20 to 50)

4:1

Sexually acquired ReA; Ct IgG positive^a, Ct IgA positive serology^b

Sexually acquired ReA;

Sexually acquired ReA; Ct IgG positive serology^a; Ct IgA positive serology^b; B27+^d

Sexually acquired ReA; Ct IgA positive serology^b; Ct positive PCR^c; B27+^d

Sexually acquired ReA

UA (n = 9)

25 (2 to 60)

40 (22 to 59)

5:4

RA (n = 7)

66 (12 to 228)

44 (39 to 53)

2:5

OA (n = 6)

14 (12 to 24)

58 (44 to 70)

5:1

^aSerology was determined for Ct-IgG antibodies by Micro Immunofluorescence assay as described by Wang and Grayston 29. ^bSerology was determined for Ct-IgA antibodies by ELISA (Labsystems, Hilsinki, Finland). ^cChlamydia PCR in genital swabs was determined by Cobas Amplicor PCR assay (Roche Diagnostics Molecular Systems, Inc, CA, USA). ^dHLA-B27 positivity was determined using a microcytotoxicity assay.

Ct = Chlamydia trachomatis; RA = rheumatoid arthritis; ReA = reactive arthritis; OA = osteoarthritis; UA = undifferentiated arthritis.

Automated DNA extraction from synovial fluid and broad-range PCR amplification of 16S rRNA genes, cloning and sequencing of bacterial DNA

For 10 minutes, 500 μl of SF were centrifuged at 10,000 g. The whole SF cell pellet was subjected to DNA extraction using the MagNA Pure system (Roche Molecular Biochemicals, Meylan, France). Prior to the MagNA Pure extraction, 500 μl of lysis buffer (200 mM sodium chloride, 20 mM Tris hydrochloric acid, pH 8, 50 mM ethylenediaminetetraacetic acid and 1% SDS) and 25 μl of proteinase K (10 mg/ml) (Sigma, St Louis, MO, USA) were added to the SF cell pellet.

Bacterial 16S rDNA fragments were amplified from SF extracted DNA by broad-range PCR amplification using universal bacterial 16S rRNA gene-specific oligonucleotide primers Bac08 and Uni1390, as previously described 15. Products of the expected size (approximately 1400 bp) were inserted into a vector using a cloning kit (pGEM-T vector; Promega, Madison, WI, USA). Sequencing and 16S rDNA analysis were performed as previously described 15.

Data analysis

Statistical analysis was performed using SPSS 11.0 software (SPSS; Chicago, IL, USA). A P < 0.05 was considered statistically significant.

Results

PCR positivity by the broad-range PCR amplification system

PCR was positive in 20 of the 27 (74.10%) patients indicating the presence of bacterial 16S rDNA within the synovial samples. Of note, PCR was positive in five out of five (100%) patients with ReA and also in nine out of nine (100%) patients with UA. In the control group, bacterial 16S rDNA was amplified in three out of seven (43%) RA patients and in three of six (50%) OA patients. Accordingly, the presence of bacterial DNA within the SF samples was significantly higher in ReA and UA patients as compared with RA and OA patients (14 out of 14 (100%) vs 6 of 13 (46.2%); P = 0.006).

At least 24 individual clones were selected from each PCR product and sequenced. A summary of the total number of sequences analyzed in each patient is depicted in Table 2. Only full-length (1300 to 1400 bp) sequences of high quality were analyzed in detail. Most bacterial sequences had at least 97% sequence similarity with any known cultivated or uncultured bacteria. The percentage of similarity to the best fit sequence in the database, the accession number, and the sequence length of each probe are listed in Table 3.

Table 2

Details of bacterial species-derived DNA sequences identified in each patient*

Patient

Total number of bacterial DNA sequences

DNA sequences identified in each patient

ReA

10 × Stenotrophomonas maltophilia, 7 × Shigella species, 6 × Propionibacterium acnes, 3 × Ralstonia species, 3 × Shigella sonnei, 3 × uncultured bacterium, 2 × Bradyrhizobium elkanii, 1 × uncultured γ proteobacterium, 1 × uncultured γ proteobacterium, 2 × uncultured Sphingobacterium species, 1 × Aranicola species, 1 × Agrobacterium species, 1 × Burkholderia species, 1 × Escherichia coli, 1 × Paenibacillus humicus, 1 × Pantoea ananatis, 1 × uncultured β proteobacterium.

13 × Stenotrophomonas maltophilia, 4× Shigella species, 3 × Aranicola species, 3 × Ralstonia species, 2 × β proteobacterium, 2 × γ proteobacterium, 2 × uncultured β proteobacterium, 1 × Agrobacterium species,, 1 × Bacteroidetes bacterium, 1 × Escherichia coli, 1 × Escherichia species, 1 × Flavobacterium mizutaii, 1 × Pantoea ananatis, 1 × Paenibacillus humicus, 1 × Paenibacillus species, 1 × Propionibacterium acnes, 3 × Shigella sonnei 1 × Streptococcus mitis, 1 × swine manure bacterium, 1 × uncultured α proteobacterium, 1 × uncultured Bacteroidetes bacterium, 1 × uncultured Flavobacterium species, 1 × uncultured soil bacterium.

10 × Shigella species, 9 × Stenotrophomonas maltophilia, 3 × Aranicola species, 3 × uncultured bacterium,, 2 × Escherichia species, 2 × Paenibacillus humicus, 2 × Streptococcus mitis, 1 × Achromobacter xylosoxidans, 1 × Bradyrhizobium elkanii, 1 × Bacteroidetes bacterium, 1 × Flavobacterium mizutaii, 1 × γ proteobacterium, 1 × Propionibacterium acnes, 1 × Shigella sonnei, 1 × Streptococcus mitis, 1 × uncultured Flavobacterium species, 1 × uncultured Methylococcaceae bacterium, 1 × uncultured Bacteroidetes bacterium

11 × Stenotrophomonas maltophilia, 6 × γ proteobacterium, 5 × Shigella species, 5 × Shigella sonnei, 3 × Aranicola species, 3 × Comamonas testosteroni, 3 × uncultured bacterium, 2 × Escherichia species, 2× Propionibacterium acnes, 2 × uncultured Flavobacterium species, 2 × uncultured β proteobacterium, 1 × Alcaligenes faecalis, 1 × Flavobacterium mizutaii, 1 × Paenibacillus humicus 1 × Pelomonas saccharophila, 1 × Pseudomonas species.

5 × Escherichia coli, 4 × Stenotrophomonas maltophilia, 3 × Escherichia coli, 2 × Pseudomonas species, 2 × Shigella species, 2 × Sphingomonas faeni, 1 × Alcaligenes faecalis, 1 × β proteobacterium, 1 × Comamonas testosteroni, 1 × Diaphorobacter species, 1 × Delftia acidovorans, 1 ×Flavobacterium mizutaii, 1 × γ proteobacterium, 1 × uncultured bacterium, 1 × uncultured β proteobacterium, 1 × Pantoea ananatis, 1 × Propionibacterium acnes, 1 × Ralstonia species, 1 × Shigella sonnei,

13 × Stenotrophomonas maltophilia, 9 × Shigella species, 7 × γ proteobacterium, 2 × uncultured bacterium, 2 × uncultured Veillonella species, 1 × Alcaligenes faecalis, 1× Agrobacterium species, 1 × Escherichia species, 1 × Streptococcus thermophilus, 1 × Flavobacterium mizutaii, 1 × Propionibacterium acnes, 1 × uncultured β proteobacterium, 1 × uncultured bacterium, 1 × uncultured candidate division OP10 bacterium, 1 × Pantoea ananatis, 1 × Shigella sonnei, 1 × unidentiefied bacterium, 1 × uncultured Flavobacterium species, 1 × uncultured Sphingobacterium species.

8 × Stenotrophomonas maltophilia, 3 × Shigella species, 2 × Comamonas testosteroni, 2 × Paenibacillus humicus, 2 × Ralstonia species, 2 × uncultured bacterium, 2 × uncultured β proteobacterium, 2 × uncultured Flavobacterium species, 1 × Burkholderia species, 1 × Bradyrhizobium elkanii, 1 × Bacteroidetes bacterium, 1 × Escherichia coli, 1 × Flavobacterium mizutaii, 1 × γ proteobacterium, 1 × Shigella sonnei

15 × Stenotrophomonas maltophilia, 5 × Shigella species, 3 × Escherichia species, 3 × Paenibacillus humicus, 2 × uncultured bacterium, 2 × uncultured β proteobacterium, 1 × Alcaligenes faecalis, 1 × Aranicola species, 1 × Bradyrhizobium elkanii, 1 × Flavobacterium mizutaii, 1 × γ proteobacterium, 1 × Ralstonia species, 1 × Staphylococcus pasteuri, 1 × Streptococcus mitis, 1 × Streptococcus pneumoniae, 1 × Streptococcus species, 1 × uncultured candidate division OP10 bacterium, 1 × uncultured Streptococcus species, 1 × uncultured Veillonella species.

11 × Aquabacterium commune, 8 × Shigella species, 3 × Aranicola species, 3 × Paenibacillus humicus, 2 × Alcaligenes faecalis, 2 uncultured bacterium, 1 × β proteobacterium, 1 × Comamonas testosteroni, 1× Escherichia species, 1 × Kocuria species, 1 × Pseudomonas species, 1 × Paracoccus species, 1 × Streptococcus thermophilus, 1 × Streptococcus species, 1 × uncultured β proteobacterium

13 × Stenotrophomonas maltophilia, 4 × Shigella species, 3 × γ proteobacterium, 3 × Shigella sonnei, 1 × Aranicola species, 1 × Alcaligenes faecalis, 1× Aeromonas species, 1× Burkholderia species, 1 × Bradyrhizobium elkanii, 1 × Comamonas testosteroni, 1 × Capnocytophaga sputigena 1 × Enterococcus faecium, 1× Escherichia species, 1 × Enterobacter hormaechei, 1 × Pseudomonas poae, 1 × Pantoea ananatis, 1 × Paenibacillus humicus, 1 × Rhodococcus species, 1 × Ralstonia species, 1 uncultured bacterium, 1 × uncultured γ proteobacterium, 1 × uncultured Flavobacterium species.

6 × Stenotrophomonas maltophilia, 5 × Shigella species, 4 × γ proteobacterium, 4 × Ralstonia species, 3 × Escherichia coli, 2 × Bradyrhizobium elkanii, 2 × Comamonas testosteroni, 2 × uncultured bacterium, 1 × Achromobacter xylosoxidans, 1 × Alcaligenes faecalis, 1 × Bacteroidetes bacterium, 1 × Flavobacterium mizutaii, 1 × Rhizobium radiobacter, 1 × Rhodococcus species, 1 × uncultured β proteobacterium, 1 × uncultured organism.

4 × Shigella species, 4 × Stenotrophomonas maltophilia, 3 × Alcaligenes faecalis, 2 × Shigella sonnei, 2 × uncultured β proteobacterium, 2 × uncultured bacterium, 1 × Aminobacter aminovorans, 1 × Achromobacter xylosoxidans, 1 × Paenibacillus humicus, 1 × Ralstonia species, 1 × uncultured organism.

12 × Stenotrophomonas maltophilia, 3 × Shigella species, 3 × uncultured bacterium, 2 × Bacillus niacini, 2 × Ralstonia species, 1 × β proteobacterium, 1 × Bradyrhizobium japonicum, 1 × Burkholderia cepacia, 1 × Corynebacterium durum, 1 × Comamonas testosteroni, 1 × Flavobacterium mizutaii, 1 × γ proteobacterium, 1 × uncultured β proteobacterium, 1 × uncultured Sphingobacterium species, 1 × Mycobacterium aubagnense, 1 × Shigella sonnei., 1 × Sphingomonas species, 1 × Sphingomonas species, 1 × Pseudomonas poae, 1 × Pantoea ananatis.

10 × Stenotrophomonas maltophilia, 7 × Ralstonia species, 3 × Comamonas testosteroni, 3 × uncultured bacterium, 2 × γ proteobacterium, 1 × Escherichia species, 1 × Flavobacterium mizutaii, 1 × Methylomicrobium species, 1 × Paenibacillus humicus, 1 × Pantoea ananatis, 1 × Photorhabdus luminescens, 1 × Streptococcus pneumoniae, 1 × uncultured Sphingobacterium species, 1 × uncultured Flavobacterium species, 1 × uncultured β proteobacterium, 1 × uncultured Firmicutes bacterium.

10 × Stenotrophomonas maltophilia, 3 × uncultured Flavobacterium species, 2 × Comamonas testosteroni, 2 × uncultured bacterium, 1 × Aranicola species, 1 × Flavobacterium mizutaii, 1 × Paenibacillus humicus, 1 × Shigella species, 1 × Staphylococcus cohnii, 1 × Sphingobacterium thalpophilum, 1 × uncultured Sphingobacterium species.

5 × uncultured β proteobacterium, 4 × uncultured Flavobacterium species, 2 × uncultured Sphingobacterium species, 1 × Comamonas testosterone, 1 × uncultured bacterium.

3 × Escherichia species, 2 × uncultured bacterium, 1 × Alcaligenes faecalis, 1 × Comamonas testosteroni, 1 × Shigella species.

4 × Stenotrophomonas maltophilia, 4 × Shigella species, 2 × Flavobacterium mizutaii, 2 × uncultured Flavobacterium species, 2 × uncultured Sphingobacterium species, 1 × Acinetobacter junii, 1 × Escherichia coli O157, 2 × uncultured bacterium.

5 × Stenotrophomonas maltophilia, 3 × Shigella species, 3 × uncultured bacterium, 1 × Alcaligenes faecalis, 1 × Comamonas testosteroni, 1 × Flavobacterium mizutaii, 1 × Paenibacillus humicus, 1 × Ralstonia species.

4 × Stenotrophomonas maltophilia, 3 × Shigella species, 3 × uncultured bacterium, 1 × Achromobacter xylosoxidans, 1 × Alcaligenes faecalis, 1 × Bacillus cereus, 1 × Escherichia species, 1 × Rothia mucilaginosa, 2 × uncultured β proteobacterium

OA = osteoarthritis; RA = rheumatoid arthritis; ReA = reactive arthritis; UA = undifferentiated arthritis.

Table 3

Bacterial species identified by sequencing of cloned 16S rDNA

Bacterium-derived DNA identified in SF samples

Number of patients in whom bacterial DNAs were detected

Accession number^a

Length of the sequence^b

% Similarity^c

Bacteria identified in ReA and UA patients (n = 69)

Bacteria previously detected in arthritis

Aeromonas species

(1 UA)

[EMBL:AF099027]

1400

97.77

Burkholderia species

(1 ReA + 1 UA)

[EMBL:AY769903]

1391

99.13

Burkholderia species

(1 UA)

[EMBL:AY134849]

1388

99.71

Burkholderia cepacia

(1 UA)

[EMBL:AY946010]

1390

99.89

Escherichia coli

(1 ReA)

[EMBL:CP000243]

1400

99.14

Escherichia coli

(2 ReA)

[EMBL:U00096]

1400

100.00

Escherichia coli

(1 ReA + 2 UA)

[EMBL:V00348]

1400

99.50

Methylomicrobium species

(1 UA)

[EMBL:AF194538]

1380

98.83

Propionibacterium acnes

(5 ReA + 1 UA)

[EMBL:AB108477]

1386

99.05

Pseudomonas species

(1 ReA)

[EMBL:DQ213044]

1386

99.06

Pseudomonas species

(1 ReA)

[EMBL:AY014811]

1395

99.06

Pseudomonas species

(1 UA)

[EMBL:AM409368]

1396

99.21

Paracoccus species

(1 UA)

[EMBL:AY745834]

1320

98.92

Rhodococcus species

(2 UA)

[EMBL:AF420412]

1374

97.29

Shigella sonnei

(5 ReA + 5 UA)

[EMBL:CP000038]

1400

99.71

Shigella sonnei

(1 UA)

[EMBL:X96964]

1389

99.78

Sphingomonas species

(1 UA)

[EMBL:AB110635]

1341

99.85

Sphingomonas species

(1 UA)

[EMBL:AF385529]

1341

100.00

Streptococcus species

(1 UA)

[EMBL:AF316593]

1400

98.55

Streptococcus species

(1 UA)

[EMBL:AF385523]

1400

99.28

Streptococcus mitis

(1 ReA)

[EMBL:AJ295848]

1400

97.92

Streptococcus mitis

(1 ReA)

[EMBL:AJ617805]

1322

98.71

Streptococcus mitis

(1 ReA)

[EMBL:AF003929]

1396

99.36

Streptococcus mitis

(1 ReA + 1 UA)

[EMBL:AY518677]

1404

99.86

Streptococcus pneumoniae

(2 UA)

[EMBL:AM157442]

1400

98.64

Streptococcus thermophilus

(2 UA)

[EMBL:AY188354]

1397

99.57

Bacteria not previously detected in arthritis but detected in human infection

Agrobacterium species

(2 ReA + 1 UA)

[EMBL:AY775177]

1344

99.40

Bradyrhizobium elkanii

(2 ReA + 4 UA)

[EMBL:AY904749]

1347

99.40

Capnocytophaga sputigena

(1 UA)

[EMBL:AF133536]

1378

98.96

Corynebacterium durum

(1 UA)

[EMBL:AF537593]

1391

99.58

Delftia acidovorans

(1 ReA)

[EMBL:AB020186]

1309

97.63

Enterococcus faecium

(1 UA)

[EMBL:EF533988]

1396

99.42

Enterobacter hormaechei

(1 UA)

[EMBL:AY995561]

1400

99.70

Kocuria species

(1 UA)

[EMBL:AY864652]

1387

99.85

Mycobacterium aubagnense

(1 UA)

[EMBL:AY859683]

1372

99.27

Pantoea ananatis

(3 ReA + 4 UA)

[EMBL:DQ133546]

1400

98.80

Photorhabdus luminescens

(1 UA)

[EMBL:AY444555]

1333

99.85

Pseudomonas poae

(2 UA)

[EMBL:AJ492829]

1386

98.99

Rhizobium radiobacter

(1 UA)

[EMBL:AJ389902]

1300

99.67

Staphylococcus pasteuri

(1 UA)

[EMBL:AJ717376]

1411

99.86

Bacteria not previously detected in humans

Aquabacterium commune

(1 UA)

[EMBL:AF035054]

1416

99.70

Aminobacter aminovorans

(1 UA)

[EMBL:AJ011759]

1344

98.72

Bacillus niacini

(1 UA)

[EMBL:AB021194]

1400

99.14

Bradyrhizobium japonicum

(1 UA)

[EMBL:AB072418]

1200

97.81

Diaphorobacter species

(1 ReA)

[EMBL:DQ294626]

1387

99.78

Pelomonas saccharophila

(1 ReA)

[EMBL:AB021407]

1381

99.42

Paenibacillus species

(1 ReA)

[EMBL:AM162345]

1385

99.35

Sphingomonas faeni

(1 ReA)

[EMBL:AJ429239]

1340

97.82

Uncultured bacteria

Bacteroidetes bacterium

(2 UA + 1 ReA)

[EMBL:AY395022]

1394

96.11*

Bacteroidetes bacterium

(1 ReA)

[EMBL:DQ195837]

1300

98.39

β Proteobacterium

(2 ReA + 2 UA)

[EMBL:AY162033]

1396

99.57

γ Proteobacterium

(4 ReA + 6 UA)

[EMBL:AY162042]

1399

99.00

γ Proteobacterium

(1 ReA + 4 UA)

[EMBL:AY162068]

1400

99.50

swine manure bacterium

(1 ReA)

[EMBL:AY167969 ]

1395

99.21

Uncultured α proteobacterium

(1 ReA)

[EMBL:AF445680]

1342

97.00

Uncultured Bacteroidetes bacterium

(2 ReA)

[EMBL:AY921801]

1384

97.04

Uncultured bacterium

(2 UA)

[EMBL:AY958813]

1388

99.88

Uncultured β proteobacterium

(1 ReA + 1 UA)

[EMBL:AF445700]

1372

99.78

Uncultured β proteobacterium

(1 UA)

[EMBL:DQ316806]

1391

97.46

Uncultured candidate division OP10 bacterium

(2 UA)

[EMBL:AF418946]

1362

90.29*

Uncultured Firmicutes bacterium

(1 UA)

[EMBL:EF071401]

1400

99.57

Uncultured γ Proteobacterium

(1 UA)

[EMBL:AF324537]

1396

99.96

Uncultured γ Proteobacterium

(1 ReA)

[EMBL:AJ318146]

1400

97.63

Uncultured γ Proteobacterium

(1 ReA)

[EMBL:AY770720]

1393

96.91*

Uncultured Methylococcaceae bacterium

(1 ReA)

[EMBL:EF019533]

1398

97.55

Uncultured organism

(2 UA)

[EMBL:DQ395839]

1392

99.85

Uncultured soil bacterium

(1 ReA)

[EMBL:DQ297948]

1340

99.85

Uncultured Streptococcus species

(1 UA)

[EMBL:AY256519]

1400

99.21

Uncultured Veillonella species

(2 UA)

[EMBL:AM157449]

1400

99.37

Bacteria identified in control group (RA and OA patients; n = 10)

Bacteria previously detected in arthritis (in joint)

Acinetobacter junii

(1 OA)

[EMBL:AB101444]

1387

99.28

Bacillus cereus

(1 OA)

[EMBL:AB247137]

1399

98.36

Rothia mucilaginosa

(1 OA)

[EMBL:DQ409140]

1375

98.69

Staphylococcus cohnii

(1 RA)

[EMBL:AJ717378]

1399

99.57

Bacteria not previously detected in arthritis but detected in human infection

Escherichia coli O157

(1 OA)

[EMBL:AE005174]

1394

97.56

Bacteria not previously detected in humans

Sphingobacterium thalpophilum

(1 RA)

[EMBL:AJ43817]

1388

94.02*

Uncultured bacteria

Uncultured bacterium

(1 OA)

[EMBL:DQ800655]

1394

97.99

Uncultured bacterium

(1 RA)

[EMBL:AY958855]

1388

99.78

Uncultured bacterium

(1 RA)

[EMBL:AY958896]

1380

99.57

Uncultured bacterium

(1 RA + 1 OA)

[EMBL:DQ818800]

1398

99.57

Common bacteria^d(n = 20)

Bacteria previously detected in arthritis

Achromobacter xylosoxidans

(1 ReA + 2 UA+ 1 OA)

[EMBL:AF439314]

1389

99.50

Alcaligenes faecalis

(2 ReA + 6 UA + 2 OA + 1 RA)

[EMBL:AY548384]

1395

99.70

Comamonas testosterone

(1 ReA + 4 UA +1 OA + 3 RA)

[EMBL:AB007996]

1390

99.35

Comamonas testosterone

(2 ReA + 3 UA + 1 OA)

[EMBL:M11224]

1390

98.05

Escherichia species

(3 ReA + 5 UA + 1 OA + 1 RA)

[EMBL:DQ337503]

1400

99.86

Flavobacterium mizutaii

(4 ReA + 6 UA + 2 OA + 1 RA)

[EMBL:AJ438175]

1385

94.00*

Shigella species

(5 ReA + 8 UA + 3 OA + 2 RA)

[EMBL:DQ337523]

1399

99.70

Stenotrophomonas maltophilia

(5 ReA + 8 UA + 3 OA + 1 RA)

[EMBL:AJ293470]

1396

99.75

Stenotrophomonas maltophilia

(5 ReA + 8 UA + 3 OA + 1 RA)

[EMBL:AB294557]

1396

99.86

Bacteria not previously detected in arthritis but detected in human infection

Aranicola species

(4 ReA + 3 UA + 1 RA)

[EMBL:AM398227]

1400

99.64

Ralstonia species

(3 ReA + 7 UA + 1 OA)

[EMBL:AB045276]

1387

99.86

Bacteria not previously detected in humans

Paenibacillus humicus

(4 ReA + 6 UA +1 OA + 1 RA)

[EMBL:AM411528]

1399

98.60

Uncultured bacteria

Uncultured bacterium

(4 ReA + 4 UA + 1 OA)

[EMBL:DQ818781]

1394

98.90

Uncultured bacterium

(5 ReA + 4 UA + 2 OA + 2 RA)

[EMBL:AY838480]

1389

94.00*

Uncultured bacterium

(2 ReA + 3 UA + 1 OA + 2 RA)

[EMBL:AB076874]

1389

94.00*

Uncultured bacterium

(1 ReA +1 OA)

[EMBL:AY838458]

1382

99.64

Uncultured bacterium

(1 UA + 1 OA)

[EMBL:DQ824599]

1398

99.57

Uncultured β proteobacterium

(4 ReA + 6 UA + 1 OA + 1 RA)

[EMBL:DQ366010]

1384

99.71

Uncultured Flavobacterium species

(3 ReA + 5 UA + 1 OA + 2 RA)

[EMBL:DQ366085]

1300

97.20

Uncultured Sphingobacterium species

(1 ReA + 3 UA + 1 OA + 2 RA)

[EMBL:AB076874]

1389

94.16*

Number in brackets after species names indicate the number of patient set from whom bacteria were detected. ^aAccession number of the bacterial species in the EMBL database. ^bLength of alignment on which the 16S rDNA inserted sequence and the corresponding sequence in the database are similar. ^cIn the '% similarity' column, asterisks indicate highlight instances where the % similarity is below 97%. ^dThe 'Common bacteria' row shows the bacteria identified in ReA, UA, RA, and OA patients.

Bacterial species detected only in SF samples and not in ST samples from our previous study 15, are indicated in bold.

OA = osteoarthritis; RA = rheumatoid arthritis; ReA = reactive arthritis; SF = synovial fluid; UA = undifferentiated arthritis.

Bacterial 16S rDNA sequences identified in synovial fluid samples

Each patient's SF samples contained a diverse range of bacterial DNA-related species (Table 2). The majority of pathogens across all groups were identified as Shigella species and Stenotrophomonas maltophilia. DNA from a total of 69 various individual bacterial species were detected in SF samples from ReA and UA patients, whereas only 10 different bacterial DNA were found in SF samples from RA and OA patients. In addition, DNA from 20 bacterial species was detected in both the study and the control groups.

In ReA and UA patients, apart from Shigella sonnei DNA, there was no other DNA-derived bacteria so far described to trigger ReA. There were sequences from commensal bacteria, in particular those from the skin or the intestinal tract (Propionibacterium acnes, Escherichia coli, and other coliform bacteria). We also detected additional species previously assigned to the Pseudomonas genus such as DNA of Pseudomonas poae, Delftia acidovorans, and Burkholderia cepacia. A detailed sequence analysis of the PCR-positive samples of ReA and UA patients revealed a number of DNA of bacteria that have previously been described in human infections but not in arthritis, including Rhizobium radiobacter, Pantoea ananatis, and Capnocytophaga sputigena. Except for P. ananatis, these DNA sequences were observed in only one individual (Table 3)

DNA products from environmental bacteria previously detected in arthritis, such as Achromobacter xylosoxidans, Alcaligenes faecalis, and Flavobacterium mizutaii were commonly found in both the study and control groups. DNA of Aranicola species bacterium rarely described in humans but not associated with arthritis was common in patients with ReA, UA, and RA and was detected in more than one case (Table 3).

Some clones fell into environmental species not previously reported in human infections including DNA of Paenibacillus humicus, Bacillus niacini, and Diaphorobacter species, and other bacteria, which have not yet been cultured (Table 3).

DNA from the candidate division OP10 bacterium was detected in SF samples of only two patients with UA. These bacterial DNA sequences have not been previously characterized by rDNA sequencing because they exhibit less than 97% similarity to known database sequences. We could find no clear association between the presence of a particular bacterial DNA and clinical symptoms.

Discussion

In the present study we used broad-range PCR amplification, cloning, and sequencing of the full-length 16S rDNA from a wide variety of bacterial species in the SF samples of all patients studied. Only a few studies have been conducted to detect and identify the bacterial DNA communities in SF samples of patients with ReA, UA, and others arthropathies. In these studies, only short fragments of DNA were amplified allowing solely the determination of the bacterial genus but not the identification of the species level. This is, to our knowledge, the first study using the full-length 16S rRNA gene as a target for broad-spectrum PCR to detect bacterial DNA in SF samples allowing the identification of the species level.

Sequence analysis of the PCR-positive samples revealed the presence of a wide spectrum of bacterial DNA in SF samples of all studied patients. False positivity due to contamination poses a problem when broad-range PCR targeting the 16S rRNA is used 202122. However, our recently published study outlines extensive measures, which were also taken in the present study in order to avoid any contamination 15. From a practical point of view, it is important to notice that we did not surgically incise the skin at the site of the aspiration thus avoiding contamination by the skin flora but our results are rather similar to those obtained by others who took this precaution 14. PCR and extraction controls consistently yielded negative results indicating that the PCR products detected in positive samples are most likely derived from the bacterial rRNA genes actually present in SF cells. In addition, the sequences obtained varied between patients and DNA from several organisms has also been identified in arthritic human joints in independent laboratories 1213141718, suggesting that their presence is unlikely to be a consequence of contamination.

The most common sequences of species found in SF samples of all patients (e.g. A. xylosoxidans, A. faecalis, F. mizutaii, and S. maltophilia) were also seen in the previous study 15, except the DNA from Aranicola species implying that they might be opportunistic colonizers of inflamed joints. S. maltophilia DNA was identified most frequently. This organism is an opportunistic pathogen and has previously been detected in arthritic knee joints 121517. Of note, other bacterial DNA have been described in human infections but not so far in arthritis were identified in the SF samples of our arthritic patients. Some of them are detected only in SF samples but not in their matched ST samples, such as R. radiobacter and Pantoae ananatis. Recently, we reported that DNA derived from uncultured bacteria and from environmental organisms that have not been previously detected in human samples could also be demonstrated in ST samples 15. Similarly, in SF samples such bacterial DNA was detected including Aminobacter aminovorans, B. niacini, Diaphorobacter species, and uncultured candidate division OP10 bacterium. Several studies have also detected unsuspected uncultured and/or cultured bacteria not considered as human pathogens in arthritic joints but they were found by sequencing short DNA fragments 12131416. Thus, the identification of such bacterial species after cloning and near-full length 16S DNA sequencing might be of interest and should be pursued. However, their presence in the joint can not provide definite evidence of their replication or a functional role in arthritis.

Our results also confirmed the presence of E. coli sequences in SF samples as previously found in ST samples of arthritis patients 15. This could indicate the ability of E. coli DNA to colonize inflamed joints; the gut in different patients would be expected to contain a material derived from a range of E. coli 'subspecies' 232425.

Our analysis of SF samples and their matched ST samples confirmed a wide spectrum of bacterial DNA-related species detected in each individual patient. Accordingly, a significant correlation was found between the diversity of bacterial species detected in SF and matched ST at the patient level (r = 0.522, P = 0.018). However, they revealed a different profile in regard to their known potential of triggering ReA in either SF or ST samples. Thus, the Shigella flexneri sequences were not detected in any of the SF samples whereas S. sonnei 16 rDNA sequences were detected more frequently in SF samples of five ReA and five UA patients as compared with the ST samples of one ReA and one UA patient 15. DNA of Shigella species was also prevalent in SF samples as demonstrated previously in ST samples 15, but it was more frequently detected in SF samples (five ReA, eight UA, two RA, and three OA in SF vs two ReA, four UA, two RA, and one OA in ST samples). Although S. sonnei and Shigella species were detected more frequently in SF than in ST samples, the difference was statistically not significant.

We detected DNA from Shigella species in our cohort of patients with various forms of arthritis and S. sonnei, known to trigger ReA, only in ReA and UA samples. Shigella DNA positive patients had no clinical signs of previous intestinal infection with an enteric organism. These patients may have been asymptomatic, or the preceding gastrointestinal symptoms may have been mild and overlooked by the patients 3. It is possible that enteric organisms may migrate from asymptomatic primary sites of the infection to the synovial compartment 3. Most Shigella ReA caused by S. sonnei are sporadic cases 26. The most recent published case of S. sonnei related ReA was attributed to sexual transmission of the pathogen 27. In our study, we detected S. sonnei DNA in five ReA patients presenting with an urogenital infection, which is consistent with the possibility that this species could be related to sexual transmission.

A composition of a mixture of bacterial nucleic acids was common in our cohort of patients with various forms of arthritis, as has been described in previous studies 1314151718. The bacterial DNA might be incorporated by macrophages, which are disseminated by the circulation and reach the joint due to an increased cellular recruitment. As opposed to a single organism, such mixtures may increase the risk of triggering an immune response finally culminating in synovitis. However, genetic susceptibility factors of the host are also playing an important role particularly in persistent infections 1028. As mixtures of bacterial nucleic acids are also detectable in the SF samples from patients with RA and OA, we cannot exclude that this may represent a normal 'background' phenomenon not necessarily causing synovitis 1017. Another limitation of our study lies in the fact that we could not detect Chlamydia trachomatis DNA or other common bacteria known to trigger posturethritic arthritis.

Conclusions

Our study provides a valuable overall picture of the bacterial DNA environment present in the SF of the actively inflamed joints of arthritis patients. Characterization of the DNA reveals a wide spectrum of organisms so far not known to be present in human infections, not known to be present in inflamed joints of arthritis patients, and not known to trigger ReA. There is also a differential bacterial colonisation and/or infection of SF and ST samples because the analysis of SF can identify a number of bacterial DNA-related species, which have not been detected in ST samples as studied earlier 15 and has helped to confirm that the composition of bacterial DNA may change over time in joint cavity.

Accordingly, the analysis of SF or ST samples from different arthropathies patients by broad-range PCR is essentially capable of characterising the bacterial DNA environment present in joint cavity. As synovial biopsy is a difficult act, SF is well practical for such purpose.

Abbreviations

OA: osteoarthritis; PCR: polymerase chain reaction; RA: rheumatoid arthritis; ReA: reactive arthritis; SF: synovial fluid; ST: synovial tissue; UA: undifferentiated arthritis.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

MS performed the experimental work, analyzed the data, and wrote the manuscript. RG conceived of the study, performed the design and coordination of the study, analyzed the data, and revised the manuscript. HF, MY, SB, NB, and SS made pathological diagnosis, conducted sampling procedures, and performed clinical and rheumatological data analyses. BJ and JS participated in the design and coordination of the study, and drafted the manuscript. MR has assisted in writing the manuscript. AH and AS analyzed microbiological and sequencing data, and revised the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We thank Sebastien Chaussonneri and Sonda Guermazi (CEA-Genoscope) for help with sequence analysis and for technical assistance. We also thank Ilhem Cheour (Tunis), Nihel Meddeb (Tunis), Mohamed Moalla (Tunis), and Imed kolsi (Sfax) for providing patient synovial samples. This project was supported by grants from the Ministry of research and development of Tunisia with participation of funds from CEA-Genoscope-Evry-France.

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