Comparison of the faecal microflora of patients with ankylosing spondylitis and controls using molecular methods of analysis

S. Stebbings1,, K. Munro2, M. A. Simon2, G. Tannock2, J. Highton3, H. Harmsen4, G. Welling4, P. Seksik5, J. Dore5, G. Grame5 and A. Tilsala-Timisjarvi6

1 Department of Rheumatology, Southmead Hospital, Bristol, UK,
2 Department of Microbiology, University of Otago, Dunedin,
3 Department of Medical and Surgical Sciences, University of Otago, New Zealand,
4 Department of Medical Microbiology, University of Groningen, The Netherlands,
5 INRA, Jouy-en-Josas, France and
6 Department of Biology, University of Oulu, Finland


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Objectives. To determine whether differences within the complex intestinal microflora can be demonstrated between patients with ankylosing spondylitis (AS) and healthy individuals.

Methods. The composition of the faecal microflora of 15 ankylosing spondylitis patients and 15 matched controls was determined using a variety of nucleic acid-based methods, including denaturing gradient gel electrophoresis (DGGE). Concentrations of serum antibodies reactive with intestinal bacteria were determined. T-cell proliferation responses to autologous intestinal bacteria were determined using a bioluminescent assay.

Results. DGGE demonstrated a unique and stable bacterial community in the faeces of each individual. No specific differences in colonization profiles were discernible between patients and controls. Analysis of individual bacterial groups using nucleic acid-based methods showed no differences in faecal colonization with Klebsiella pneumoniae or Bacteroides vulgatus. A significantly higher proportion of faecal samples from AS patients were found to contain sulphate-reducing bacteria compared with samples from controls (P=0.0004). Three out of five patients showed elevated T-cell proliferation responses to Bacteroides species cultured from their own faeces. The concentrations of serum immunoglobulin A (IgA) and IgM antibodies reactive with klebsiella or bacteroides cells were lower in the patient group relative to the controls.

Conclusions. By using DGGE, we have demonstrated the complexity and individuality of the human intestinal microflora and shown that this is a confounding factor in determining the possible significance of individual organisms in the pathogenesis of spondyloarthritis. Nevertheless, we demonstrated a higher prevalence of sulphate-reducing bacteria in the faeces of patients with AS. These organisms have been implicated in the pathogenesis of inflammatory bowel disease. We also detected a possible loss of immunological tolerance to autologous Bacteroides isolates in patients with AS.

KEY WORDS: Faecal microflora, Ankylosing spondylitis, Sulphate-reducing bacteria, Denaturing gradient gel electrophoresis, PCR.


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Ankylosing spondylitis (AS) is an inflammatory disease of the axial and, to a lesser extent, the peripheral joints. The sacroiliac joints are almost always affected. The majority of the patients possess the HLA-B27 gene, although other genes are likely to contribute to predisposition to the disease [1]. The involvement of the intestinal microflora in the pathogenesis of AS is indicated by the occurrence of colitis and arthritis in rats rendered transgenic for human HLA-B27 but the absence of inflammatory lesions when such animals are maintained germ-free [2]. In humans, patients with inflammatory bowel disease may also suffer from AS and, conversely, ileitis has been detected in patients suffering from spondyloarthritis in the absence of Crohn's disease or ulcerative colitis [3]. Recently, a specific gene mutation resulting in a defect of innate immunity has been identified in patients with Crohn's disease, and provides a further potential link between the immune response to the intestinal microflora and the development of disease [4, 5].

The composition of the intestinal microflora of humans suffering from inflammatory diseases of the bowel and spine has not been compared comprehensively with that of matched healthy individuals. Investigations of the intestinal microflora have been hampered by the difficulty of cultivating members of the intestinal microflora in the laboratory; indeed, even using state-of-the-art bacteriological methods about 60% of the bacterial community is currently non-cultivable [6]. Specific bacterial groups have been suggested to have importance in the pathogenesis of AS, in particular Klebsiella pneumoniae [7] and Bacteroides vulgatus [8]. It has also been suggested that Helicobacter species [9, 10] and sulphate-reducing bacteria [11] are involved in the pathogenesis of inflammatory bowel disease (IBD). New methods for the analysis of complex bacterial communities have been developed recently. These methods permit comprehensive comparisons to be made of the intestinal microflora in health and disease, as these techniques are nucleic acid-based and detect cultivable and non-cultivable bacteria [12]. We have applied these methods to the analysis of the faecal microflora in patients with AS and the detection of specific organisms previously implicated in the pathogenesis of AS and IBD.


    Methods
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 Introduction
 Methods
 Results
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 References
 
Subjects
Fifteen patients with AS were recruited from a database held at Dunedin Public Hospital. All patients were Caucasian and fulfilled the modified New York criteria for AS [13] and the European Spondyloarthropathy Study Group (ESSG) criteria for spondyloarthritis [14]. An assessment of disease severity was made on the basis of core indicators identified by van der Heijde et al. [15]. Five parameters were scored for severity: the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI); axial involvement (measured by chest expansion and the modified Schober's test); C-reactive protein; number of peripheral joints involved; and sacroiliac joint disease (graded according to the New York criteria) [15]. Patients were graded as having mild, moderate or severe disease on this basis. Median disease duration in the patient group was 14 yr. Characteristics of the patient group are given in Table 1Go. The controls were recruited from blood donors and students and were matched to patients on the basis of age and gender. Ethics approval for the study was obtained from the Otago Ethics Committee and written informed consent to participation was obtained from the subjects. Patients and controls provided three faecal samples, collected at monthly intervals. Blood samples were collected at the time of initial assessment and sera were stored at -70°C until assayed.


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TABLE 1. Clinical and laboratory correlates: patients with AS and controls

 

Analysis of the faecal microflora
Profiles of the faecal microflora were derived by polymerase chain reaction (PCR) amplification of the V2–V3 region of the bacterial 16S ribosomal RNA genes in DNA extracted from the 90 faecal samples, followed by denaturing gradient gel electrophoresis (DGGE) [6]. This method provides a rapid and comprehensive comparison of the majority of the members of the faecal microflora. Except for the detection of sulphate-reducing bacteria, the remaining examinations were carried out on a single faecal sample from each subject. Specific bacterial groups were enumerated and expressed as a proportion of the total bacterial community in faeces by fluorescent in situ hybridization (FISH) using DNA probes [16] and by hybridization of radioactively labelled DNA probes to bacterial RNA extracted from faeces [17]. A Helicobacter genus-specific DNA sequence of 375 base pairs (bp) [18] and a sequence specific to sulphate-reducing bacteria, of 396 bp [19], were detected by PCR. PCR primers (forward primer 5'-GCA GTG AAA GAG TTC TTT GG-3'; reverse primer 5'- TCT ACG AAG TGG CCG-3') that amplified a target sequence (386 bp) specific to the RNA polymerase beta subunit (rpob) of Klebsiella pneumoniae were derived and used to test faecal DNAs for the presence of these bacteria. The PCR programme was: 94°C for 3 min then 20 cycles of 94°C for 30 s, 45°C for 30 s and 72°C for 60 s, followed by 68°C for 7 min. The PCR mix contained 10 mM Tris–HCl, 2.5 mM MgCl2, 50 mM KCl, 200 µM of each deoxynucleoside triphosphate, 20 pmol of each primer, approximately 500 ng of DNA and 2.5 U Taq DNA polymerase (Boehringer, Mannheim, Germany). Klebsiella pneumoniae ATCC 13883T DNA was used as a positive control in all PCR assays using these primers.

Antibody concentrations in sera
Immunoglobulin (Ig) G, IgA and IgM antibodies reactive with whole cells of intestinal bacteria (Klebsiella pneumoniae ATCC 13883T, implicated in AS in a previous study; Campylobacter jejuni ATCC 33560T, a common cause of gastroenteritis and associated with reactive arthritis; Bacteroides vulgatus ATCC 29527, proinflammatory in experimental animal studies; Bifidobacterium catenulatum DSM 20103T, a common inhabitant of the human bowel) were quantified in microtitre plates using ELISA (enzyme-linked immunosorbent assay) by a method described previously but without glutaraldehyde fixation of cells [20].

T-cell proliferation in response to autologous Bacteroides isolates
The proliferation of CD4+ lymphocytes resulting from exposure to 2 µg of heat-killed Bacteroides cells per ml of blood was measured by the Luminetics assay (Cyclex, Columbia, MD, USA) using freshly drawn, sodium-heparinized blood from five AS patients and five matched controls. The Bacteroides cells used as the antigen had been cultured from the subject's own faeces (autologous) on Bacteroides-Bile Aesculin Agar (Difco [6]) in an anaerobic glovebox, and the cells were prepared for the assay as described previously [21]. The proliferation index was calculated from cell activation (luminescent values) measured after exposure to Bacteroides divided by cell activation measured in non-stimulated lymphocytes.


    Results
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 Methods
 Results
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 References
 
Strikingly, all 30 subjects had unique and stable bacterial community profiles generated from their faeces. Thus each subject could be differentiated from all others by the pattern of 16S rRNA gene, V2–V3 fragments in their DGGE profile (for an example see Fig. 1AGo). Moreover, the patterns did not vary significantly over the 3 month monitoring period. Because of the complexity and individuality of faecal microfloras, it was impossible to differentiate patients and controls on the basis of the composition of the whole faecal microflora (Fig. 1B, CGo).



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FIG. 1. (A) Faecal bacterial community profiles generated by PCR/DGGE. Three faecal samples collected at monthly intervals from four control subjects. Lanes 1–3, subject CT02; lanes 4–6, subject CT04; lanes 7–9, subject CT05; lanes 10–12, subject CT07. (B) Faecal bacterial community profiles of the AS patients. Lanes 1–15 show the profiles obtained from each of 15 patients. (C) Faecal bacterial community profiles of the matched controls. Lanes 1–15 show the profiles obtained from each of 15 controls.

 
Populations of bacterial groups enumerated by FISH or DNA/RNA hybridization did not differ, as proportions of the total microflora, between patients and controls (Table 2Go). Total Bacteroides values appeared lower when detected by FISH compared with the DNA/RNA method, but were not statistically different (P=0.2552 for patients, 0.5536 for controls). Bacteroides vulgatus constituted 20.0% (S.E. 5.5%) of the total Bacteroides population in patients compared with 20.5% (S.E. 5.7%) in controls.


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TABLE 2. Specific bacterial groups as proportions (%) of the total faecal microflora

 
The Helicobacter genus-specific DNA sequence did not differ in incidence of detection between patients (13/15) and controls (8/15; P=0.1110, {chi}2 test with Yates' correction). The Klebsiella pneumoniae-specific sequence was not detected in any of the patients or controls. The sulphate-reducing bacterial sequence was detected at a statistically higher incidence in the faecal samples of patients (24/43 samples tested) compared with controls (7/42 faecal samples tested; P=0.0003, {chi}2 test with Yates' correction). Four patients harboured sulphate-reducing bacteria in all three faecal samples, four patients in two out of three samples, the remaining patients being negative, or positive for one sample. In contrast, only one control subject had sulphate-reducing bacteria in two samples, the remaining controls being negative for all samples examined, or positive for a single sample. Patient numbers were too small to correlate the consistent detection of sulphate-reducing bacteria with disease severity.

Serum IgA antibodies reactive with Bacteroides vulgatus were lower in concentration in sera from patients compared with controls (Table 3Go; P=0.0345, Welch's modified t-test). Serum IgM antibodies reactive with Klebsiella pneumoniae were lower in concentration in sera from patients compared with controls (Table 3Go; P=0.0463, Welch's modified t-test). The concentration of IgG antibodies reactive with the intestinal species tested did not differ (data not shown).


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TABLE 3. Concentration of serum IgA and IgM antibodies (µg/ml) reactive with intestinal bacteria

 
Comparison of proliferation indices obtained from five patients and five controls showed that, whereas control blood samples gave responses between 1.22 and 6.33, three of the patients (AS03, AS09 and AS015) gave higher values (8.44, 8.66 and 9.41 respectively). These values were 1.3–2.1 times higher than the indices recorded for their matched controls. Patient AS03 was assessed as having severe disease and patients AS09 and AS15 as having moderate disease. Patients AS05 and AS12 had a proliferation index similar to that of controls. AS12 was assessed as having mild disease, whereas AS05 had severe AS but had been treated with sulphasalazine.


    Discussion
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 Abstract
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 Methods
 Results
 Discussion
 References
 
For the purposes of the study we felt it was important to apply rigorous criteria to ensure that all patients could be clearly defined as having AS. We therefore selected the modified New York criteria for AS. In the past, it has been noted that these criteria tend to select a group of patients with longstanding severe AS and peripheral arthritis [14]. This was also our experience.

Previous studies investigating the intestinal microflora in AS have been hampered by difficulty in culturing the anaerobic bacteria which predominate [7, 2224]. In this study, PCR/DGGE was shown to provide a rapid and practical method for analysing the bacterial communities present in faecal samples. We observed that each subject harboured a unique community profile that was remarkably stable over the 3 month period of observation. PCR/DGGE analysis has been demonstrated to detect changes in the composition of the gut microflora of experimental animals [25]. In the case of human subjects, however, the uniqueness of individual microfloras confounds easy comparison of samples from patients and controls. The uniqueness of the human microfloras observed by PCR/DGGE analysis was supported by the results obtained by other molecular methods. Wide variation was detected in the proportion of the total microflora comprised of Bacteroides, for example (patients, 5.3–42.2%; controls, 6.7–64.0%). Perturbation of the faecal microflora with regard to the major groups (Bacteroides, Eubacterium–Clostridium, Bifidobacterium) enumerated by DNA hybridization methods was not detected. All patients in the study fulfilled the criteria for AS but the presence of intestinal lesions was not ascertained by endoscopy. Future studies might aim to compare the composition of the microflora associated with inflamed and normal mucosa collected from the same patient with AS. This might help to negate the confounding influence of variation in the composition of the faecal microflora between humans. The possible influence of drug therapy when comparing patients and controls cannot be excluded. However, with one exception, all patients were taking an NSAID (non-steroidal anti-inflammatory drug) and hence any difference in colonization within the patient group is unlikely to be the result of such treatment.

We did not detect differences in the incidence of Helicobacter species, Bacteroides vulgatus or Klebsiella pneumoniae in faecal samples from patients and controls. These bacterial species were selected for investigation for the following reasons. Helicobacter hepaticus and H. bilis have been implicated in inflammatory bowel disease of experimental animal models [9, 10]. H. pylori is a recognized factor in the aetiology of gastritis and peptic ulcer in humans, but there are at least 16 described species in this genus [26]. Those that predominate in human faeces remain to be determined. Germ-free HLA-B272 microglobulin transgenic rats do not develop colitis. Ex-germ-free animals associated with a cocktail of intestinal bacterial species develop colitis, which is more marked if Bacteroides vulgatus is included in the bacterial mixture [8]. Considerable research effort has been directed toward the involvement of Klebsiella pneumoniae in the pathogenesis of AS. The reported results include the presence of K. pneumoniae in faeces during active inflammatory disease [22], elevated serum antibody concentrations reactive with K. pneumoniae in patient sera [23], and antigenic similarities between HLA-B27 and Klebsiella nitrogenase [24]. The reported studies are contradictory, however, and an unequivocal role for this species in AS has not therefore been identified [24, 27].

The sulphate-reducing bacteria can reduce inorganic sulphate to hydrogen sulphide. In marine environments, they are the main scavengers of free hydrogen [28] and are believed to carry out this function in the human bowel [11]. Experimental animal models of colitis include the feeding of degraded carageenan, rich in sulphate, to guinea-pigs. Germ-free or antibiotic-treated animals are not affected [29]. Sulphates are commonly used as preservatives in processed foods and it has been proposed that this enriches for sulphate-reducing bacteria in the bowel, with a consequent increase in the concentration of toxic sulphides [30]. In one study, 92% of ulcerative colitis patients were reported to have sulphate-reducing bacteria in their faeces compared with 50% of samples from controls [11]. The potential importance of sulphate-reducing bacteria in intestinal disease is further indicated by the demonstration of intracellular bacteria resembling Desulfovibrio in association with proliferative bowel disease in ferrets, hamsters and pigs [31]. In our study, sulphate-reducing bacteria were detected in 12 of the 15 patients and in six of the 15 controls. Particularly noteworthy, however, was the persistent detection of these bacteria in the faeces of patients rather than their transient nature (one or two positive specimens) in the faeces of controls (28% of faecal samples from patients, 8% of samples from controls). We made a preliminary identification of the sulphate-reducing bacteria detected in the faeces of four patients and two controls by sequencing the PCR products. A BLAST search [32] of GenBank gave the highest score with Desulfovibrio desulfuricans when sequences amplified from both patient and control samples were tested. We enumerated sulphate-reducing bacteria in the faeces of three patients and two controls using modified Baar's agar medium (American Type Culture Collection medium 1249). The bacteria were present in similar numbers in patient and control samples (107 to 109 per gram of faeces). It would be useful in future to include sulphate-reducing bacteria in studies with ex-germ-free HLA-B27 rats in order to assess their proinflammatory potential in a host genetically predisposed to spondyloarthritis.

The differences in concentration of antibodies reactive with intestinal bacteria between patients and controls were of interest because of previous reports of this nature [23]. We found, in contrast to some other studies, that the concentration of serum antibodies reactive with whole cells of Klebsiella pneumoniae or Bacteroides vulgatus were lower in the sera of patients compared with controls. On the other hand, we observed that the CD4+ lymphocyte proliferation index, after exposure to homologous Bacteroides cells, was greater for three out of five patients than in controls. Although this result is based on a small number of observations and is thus somewhat preliminary, it is reminiscent of the reports of Duchmann et al. [33], who derived T-cell clones from peripheral blood and colonic biopsies from IBD patients and controls and tested their proliferative responses after exposure to autologous and heterologous isolates of intestinal bacteria. Lymphocytes from patients proliferated to a greater extent after exposure to heterologous compared with autologous isolates in the case of controls, whereas proliferation was equally great against autologous and heterologous strains in the case of IBD patients. Hence disease appeared to be related to a loss of tolerance to autologous intestinal bacteria.

A brief hypothesis for the aetiopathogenesis of AS could include the following elements.

  1. An intestinal inflammatory lesion results in an increase in the permeability of the intestinal mucosa.
  2. A genetically predisposed dysfunction of the immune system in which tolerance towards certain members of the microflora is lost [4, 5] could lead to the usually predominant humoral response to the bacteria being replaced by a CD4+ lymphocyte-dominated proinflammatory response. Lymphocytes activated in the intestinal mucosa are known to have almost equal affinity for the high endothelial venules of the synovium [34].
  3. Bacterial antigens from the intestinal lumen pass through the inflamed mucosa into the systemic circulation, from where macrophages within the synovium of joints are known to scavenge circulating antigenic material in a manner similar to the reticulo-endothelial system [35]. Synovitis could result from dissemination of bacterial antigens from the intestinal lumen to the joints, as occurs in reactive arthritis. The possibility that chronic synovitis may in part be a response to bacterial antigens has been strengthened by the discovery of a surprisingly diverse flora in the synovium of patients with rheumatoid arthritis [36].
  4. An exaggerated lymphocyte response to bacterial antigens seeded to the systemic circulation or the joints may prime an inflammatory response and initiate or propagate arthritis.

The finding of increased prevalence of sulphate-reducing bacteria in patients with AS raises the question of how these organisms might be involved in the pathogenic elements outlined. One possibility is that they may have proliferated in the bowel as a result of gut inflammation. Thus, such organisms may contribute to gut inflammation, at the simplest level by the production of toxic products [11, 30]. However, at another level, the presence of the intracellular desulfovibrios in inflamed colonic mucosa in animal models may be analogous to the situation in humans, where intracellular bacteria such as Salmonella, Shigella and Yersinia are known to initiate spondyloarthritis following an acute entero-colitis [31].

In summary, our study has revealed new prospects for research in the investigation of the aetiopathogenesis of AS. In particular, examination of the proinflammatory effect of sulphate-reducing bacteria is indicated. A direct way to examine this would be in HLA-B27 transgenic rats. Two other areas of interest are indicated by the data presented. First, the microflora associated with the mucosa in patients with and without ileitis could be investigated. Secondly, a comprehensive survey of the relationship between the predominant members of the microflora and the immune system in patients with AS relative to controls would be of interest.


    Acknowledgments
 
SS, GT and JH acknowledge the support of the Arthritis Research Campaign (UK), the University of Otago Research Committee, the Otago Medical Research Foundation (Community Trust of Otago), the Lottery Grants Board and the HealthCare Otago Charitable Trust. PS and JD acknowledge financial support from the Commission of the European Communities, program FLAIR CT97-3035. AT-T received support from the Academy of Finland. The provision of the APS primer sequences by Drs H. R. Gaskins and B. Deplancke prior to their publication is gratefully acknowledged.


    Notes
 
Correspondence to: S. Stebbings, Musculoskeletal Directorate, Department of Rheumatology, Southmead Hospital, Westbury-on -Trym, Bristol BS10 5NB, UK. Back


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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Submitted 1 May 2002; Accepted 21 May 2002