Abstract Inflammatory bowel disease (IBD) arises in genetically susceptible individuals as a result of an unidentified environmental trigger, possibly a hitherto unknown bacterial pathogen. Twenty-six clinical isolates of Sutterella wadsworthensis were obtained from 134 adults and 61 pediatric patients undergoing colonoscopy, of whom 69 and 29 respectively had IBD. S. wadsworthensis was initially more frequently isolated from IBD subjects, hence this comprehensive study was undertaken to elucidate its role in IBD. Utilizing these samples, a newly designed PCR was developed, to study the prevalence of this bacterium in adult patients with ulcerative colitis (UC). Sutterella wadsworthensis was detected in 83.8% of adult patients with UC as opposed to 86.1% of control subjects (p = 0.64). Selected strains from IBD cases and controls were studied to elicit morphological, proteomic, genotypic and pathogenic differences. This study reports Scanning Electron Microscopy (SEM) appearances and characteristic MALDI-TOF MS protein profiles of S. wadsworthensis for the very first time. SEM showed that the bacterium is pleomorphic, existing in predominantly two morphological forms, long rods and coccobacilli. No differences were noted in the MALDI-TOF mass spectrometry proteomic analysis. There was no distinct clustering of strains identified from cases and controls on sequence analysis. Cytokine response after monocyte challenge with strains from patients with IBD and controls did not yield any significant differences. Our studies indicate that S. wadsworthensis is unlikely to play a role in the pathogenesis of IBD. Strains from cases of IBD could not be distinguished from those identified from controls.

Citation: Mukhopadhya I, Hansen R, Nicholl CE, Alhaidan YA, Thomson JM, Berry SH, et al. (2011) A Comprehensive Evaluation of Colonic Mucosal Isolates of Sutterella wadsworthensis from Inflammatory Bowel Disease. PLoS ONE 6(10): e27076. https://doi.org/10.1371/journal.pone.0027076 Editor: Stefan Bereswill, Charité-University Medicine Berlin, Germany Received: September 29, 2011; Accepted: October 9, 2011; Published: October 31, 2011 Copyright: © 2011 Mukhopadhya et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by a Clinical Academic Training Fellowship from the Chief Scientist Office in Scotland (CAF/08/01), which also funded the salary of RH and the Broad Medical Research Program (Grant number IBD-0178). JMT was supported by grants from the GI Research Unit, Aberdeen Royal Infirmary. The Royal Hospital for Sick Children, Glasgow IBD team is generously supported by the Catherine McEwan Foundation. RKR has received support from a Medical Research Council patient research cohorts initiative grant (G0800675) for Paediatric Inflammatory Bowel Disease Cohort and Treatment Study (PICTS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Inflammatory bowel disease (IBD) is an idiopathic inflammatory disorder that is comprised of two major phenotypes, Crohn's disease (CD) and ulcerative colitis (UC). The understanding of its aetiopathogenesis has taken rapid strides in the last decade, with current investigations focusing heavily on aberrations in host-microbe interactions at the luminal intestinal surface. Genetic defects in pathogen recognition and primary handling of microbes by the innate immune system compounded with distinct changes in the luminal microbiome or dysbiosis form the current backbone of this pathogenic hypothesis [1], [2]. Despite this, researchers in the field have been striving to identify and delineate a solitary micro-organism that can explain the initiation and perpetuation of this chronic inflammatory process. In this regard, anaerobic and microaerophilic bacteria residing in the intestinal lumen have often been the neglected species, primarily on account of the intrinsic difficulty in culturing and isolating these organisms by using traditional microbiological techniques. Molecular studies have demonstrated that a substantial proportion of bacterial species (up to 30–40% of dominant species) in patients with active IBD belong to phylogenetic groups that are unusual when compared to healthy subjects [3], [4]. With this premise in mind, our laboratory has focused on enhanced and improved bacteriological conditions for the optimum growth of microaerophilic bacteria from colonic biopsy samples [5], [6]. In our pilot studies we noted the unusual preponderance of the rare microaerophilic Gram negative bacterium Sutterella wadsworthensis from cultures of biopsy samples from patients with IBD. This unusual organism has been encountered before by Mangin et al. who used 16S rRNA gene sequencing to create molecular inventories of the dominant fecal bacteria in four CD patients and four controls. They found that bacterial species which were not commonly dominant in healthy individuals were over-represented in CD. One of these species included S. wadsworthensis, which belonged to the dominant microbiota of one of the four CD patients [7]. These bacteria were first reported when performing biochemical characterization and susceptibility testing of Campylobacter gracilis clinical isolates from patients with diverse infections of the GI tract [8]. These organisms could be differentiated from C. gracilis mainly by their bile resistance and cell wall fatty acid patterns. 16S rRNA gene sequencing confirmed that these unusual organisms were not related phylogenetically to any of the Campylobacters, including C. gracilis, with the closest taxa belonging to unrelated aerobes. This was ratified by a subsequent report which demonstrated that the majority of S. wadsworthensis strains were isolated from GI infections, only occasionally being isolated from non-abdominal specimens, and were more likely to be involved in serious infections than C. gracilis [9]. The supposition was that S. wadsworthensis was a putative human pathogen. Three other species, Sutterella stercoricanis, Sutterella morbirenis and Sutterella parvirubra belonging to the same genera have subsequently been identified from canine and human feces [10], [11]. The role of this group of bacteria has not been clearly elucidated in the aetiopathogenesis of IBD. This study has for the first time outlined the role of S. wadsworthensis in patients with IBD and performed a comprehensive phenotypic, genotypic, proteomic and pathogenetic characterization of this bacterial species, which will serve as a useful benchmark for future studies.

Methods Study subjects, specimen collection and processing Adult patients were recruited from the Department of Gastroenterology at the Aberdeen Royal Infirmary. These subjects were recruited for a previous study looking at the role of enterohepatic Helicobacter in UC [5]. A total of sixty-nine patients with a diagnosis of UC made on the basis of histology of colonoscopic biopsies were recruited and assessed. Sixty-five healthy controls were contacted prior to their index colonoscopy as part of the bowel cancer screening programme and recruited for the study if they had documented absence of both macroscopic and microscopic inflammation. Children were recruited from the Departments of Paediatric Gastroenterology, Hepatology and Nutrition at the Royal Aberdeen Children's Hospital and the Royal Hospital for Sick Children (Yorkhill), Glasgow as part of an ongoing study to investigate the role of microaerophilic colonic microbiota in de-novo paediatric IBD (Bacteria in Inflammatory bowel disease in Scottish Children Undergoing Investigation before Treatment: BISCUIT study). Twenty-nine paediatric patients with newly-presenting, treatment naïve IBD and thirty-two paediatric controls undergoing routine colonoscopy were included in this present study [12]. The extent and severity of disease was assigned using the Montreal criteria [13]. Subjects were excluded if they received antibiotics within three months prior to recruitment. Mucosal colonic biopsies were obtained during the colonoscopy procedures. One to two biopsies were used for culture work and the rest were then transferred to a −80°C freezer for storage pending DNA extraction and analysis. Ethics Ethical approval for the study was granted by the North of Scotland Research Ethics Service, UK (reference numbers 04/S0802/8 and 09/S0802/24). Written informed consent was obtained from all adult subjects and from the parents of all paediatric subjects in the study. Informed assent was also obtained from older children who were deemed capable of understanding the nature of the study. Bacterial strains Sutterella wadsworthensis strains. A total of twenty-seven S. wadsworthensis strains were used in this study. The type strain, DSM 14016 ( = ATCC 51579) was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). Another twenty-six S. wadsworthensis strains obtained during the course of this study were also examined. Other bacterial strains. Twenty-eight other bacterial strains were used in this study, obtained from international culture collections as well as from clinical and environmental sources: Campylobacter jejuni (NCTC 11351), Campylobacter upsaliensis (NCTC 11540), Campylobacter fetus (NCTC 10842), Campylobacter lari (NCTC 11352), Campylobacter coli (clinical isolate), Campylobacter concisus (clinical isolate), Helicobacter pylori (ATCC 700392), Helicobacter hepaticus (ATCC 51449), Helicobacter cholecystitis (ATCC 700242), Helicobacter canis (ATCC 51402), Helicobacter canadensis (ATCC 700968), Escherichia coli (NCIMB 12201), Pseudomonas fluorescences (clinical isolate), Pseudomonas aeruginosa (ATCC 27853), Shigella sonnei (25931 clinical isolate), Proteus mirabilis (NCTC 3177), Proteus vulgaris (NCTC 4157), Salmonella enteritidis (NCTC 12694), Salmonella typhimurium (NCIMB 13284), Staphylococus aureus (NCIMB 12702), Bacillus cereus (ATCC 10876), Enterococcus faecalis (NCIMB 13280), Enterobacter aerogenes (NCIMB 10102), Listeria monocytogenes (clinical isolate), Fusobacterium nucleotum (clinical isolate), Klebsiella pneumonia (NCIMB 13281), Acinetobacter calcoaceticus (clinical isolate), Yersinia fredericksenii (NCIMB 2124), and Aeromonas caviae (clinical isolate). Sutterella wadsworthensis growth conditions S. wadsworthensis isolates were obtained by culturing 1–2 mucosal biopsy samples on five selective plates, the details of which are listed in Table 1. Biopsies were first ground in brucella broth before the resultant suspension was added to the plates. 50 µl was added to each plate with the exception of the filtered blood plate where 200 µl was first passed through a 0.45 µm filter. Cultures were incubated in a micro-aerophilic atmosphere, comprising of 5.9% oxygen, 7.2% carbon dioxide, 3.6% hydrogen and 83.3% nitrogen at 37°C. This atmosphere was generated using Anoxomat® Atmosphere Generating System, from Mart® Microbiology b.v. (9200 JB Drachten, Netherlands). Plates were reviewed twice weekly for up to one month. Any bacterial isolate deemed Gram-negative and oxygen sensitive (by virtue of failed subculture in room air) was identified by sequencing of the 16S rRNA gene and sequence search on NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Culture media used in the study. https://doi.org/10.1371/journal.pone.0027076.t001 Genotypic Characterization DNA Extraction. Genomic DNA was extracted from the colonic mucosal biopsies using the QIAamp DNA Mini Kit (Qiagen, Crawley, UK) according to an established modification of the manufacturer's instructions, optimised in-house for colonic biopsy tissue as described previously [5]. S. wadsworthensis specific primer design. For the design of a new S. wadsworthensis -specific primer, 16S rRNA gene sequences of all the S. wadsworthensis strains and other related bacterial strains were obtained from the NCBI database (http://www.ncbi.nlm.nih.gov) for multiple alignment. Nearly full length 16S rRNA sequences of S. wadsworthensis strains isolated from clinical specimens in this study were also used for the alignment analysis. Several primer sequences were initially designed by using the Primer 3 software [14] and then the most suitable one was identified manually using the BioEdit software package (version 7.0.5.3) (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). The downloaded S. wadsworthensis specific sequences were aligned with the newly designed primer sequences and the ones that matched to the highly conserved 16S rRNA gene sequences of the target species but were variable among the other bacterial species, were selected. The 20 base pair S. wadsworthensis specific primer sequences designed (SWF and SWR) are outlined in Table 2. The primer sequences were then subjected to BLAST analysis against all the other sequences within the BLAST database to ensure that at least one of the primers did not share any identical sequence with any microorganisms other than S. wadsworthensis. PPT PowerPoint slide

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larger image TIFF original image Download: Table 2. Sequences of PCR primers designed in this study. https://doi.org/10.1371/journal.pone.0027076.t002 Optimization of S. wadsworthensis PCR assay. The specificity of the newly developed S. wadsworthensis primer pair was tested against a wide panel of bacterial species as listed earlier in the methods section. The PCR assay was tested using 40 ng of bacterial DNA in a 50 µl reaction mixture consisting of 10 pmol of each primer (SWF and SWR [Sigma-Aldrich, UK]), 1X PCR buffer (Roche, UK), 250 nM of each deoxy-nucleotide-triphosphate (Bioline, UK), 2 mM MgCl 2 (Roche, UK) and 1 U of Taq polymerase (Roche,UK). The sensitivity of the PCR assay was determined by serially diluting a known quantity of target DNA (50 ng/µl to 0.005 pg/ µl) to detect the minimum concentration that would yield a visible amplicon after gel electrophoresis. Restriction fragment length polymorphism (RFLP) analysis was also done using the 555 bp S. wadsworthensis-specific PCR products using EcoR I and Hha I enymes to confirm that the PCR products were from a single bacterial species. Sequence Analysis. Sequencing was done on an Applied Biosystems model 3730 automated capillary DNA sequencer using the S. wadsworthensis - specific primers SWF and SWR or 16S rRNA specific universal bacterial primers for the whole gene amplification of the S. wadsworthensis strains. The sequences obtained were compared to those of the National Center for Biotechnology Information GenBank database using the basic local alignment search tool (BLAST) search program. Multiple alignments and phylogenetic analysis were performed using Bioedit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) (version 7.0.5.3) and a dendogram was constructed using MEGA version 4 software [15]. The evolutionary distances were calculated according to Kimura's two-parameter model. A phylogenetic tree was inferred using the neighbor-joining algorithm, and the tree topology was statistically evaluated by 1,000 bootstrap resamplings. GenBank Sequence Submission. All 16S rRNA gene sequences derived from the sequencing of S. wadsworthensis- specific PCR products were submitted to GenBank with the accession numbers from JN664092-JN664117. Phenotypic Characterization Basic biochemical tests including Gram staining, catalase, urease and oxidase tests were conducted using conventional manual methods to characterize the isolates. The strains were further characterized using the API Campy commercial biochemical kit according to the manufacturer's instructions (BioMerieux, La Balme Les Grottes, France). The purpose of using the API kits in this study was not to identify or name the bacteria, but to use the biochemical tests to further characterize the S. wadsworthensis strains. By comparing the resultant biochemical profiles, any potential differences between the strains might be elicited. Of all the API kits available it was assumed that API Campy would yield the best results, as S. wadsworthensis is phenotypically similar to Campylobacter species. Scanning Electron Microscopy (SEM) S. wadsworthensis strains were harvested for SEM. Bacterial cells were suspended in 2% Glutaraldehyde in 0.1 M sodium Cacodylate buffer (pH 7.2) for 3 hours to be fixed. The fixed cell suspensions were then applied to glass coverslips coated with poly-L-lysine and allowed to adhere for five minutes. Coverslips were then rinsed with water and post-fixed with 1% Osmium tetroxide (OsO4) for 30 minutes. They were next rinsed with water and dehydrated through a graded ethanol series (60%, 80%, and 90%) for 10 minutes each. The sample was then left for 30 minutes in absolute ethanol with ethanol changes every 10 minutes. The coverslips were kept in Hexamethyldisilazane (HMDS) Sigma® for 10 minutes, and left to dry overnight. Next day, the specimen was coated with gold using automated sputter coater EMITECH K550 (Emitech limited, Ashford, Kent) and samples were transferred to view S. wadsworthensis using SEM. Proteomic Characterization To complement the phenotypic and genotypic methods, matrix-associated laser desorption/ionisation - time of flight mass spectrometry (MALDI-TOF MS) was performed to further characterize selected S. wadsworthensis strains. Sample preparation. A total of 11 bacterial strains were analyzed by MALDI-TOF MS: eight S. wadsworthensis isolates (4 strains obtained from healthy controls and 4 strains from IBD patients), the S. wadsworthensis type strain DSM 14016, C. jejuni and C. concisus strains. Approximately 5-10 mg (half a plastic loop) of bacterial cells were harvested from fresh blood agar plates and proteins were extracted as described by Alispahic et al. (2010) [16]. The protein extract (supernatant) was diluted, with 30% acetonitrile in 0.1% trifluoracetic acid (Applied Biosystems, UK) in water. Subsequently, 1 µl of diluted supernatant was mixed with 1 µl of matrix solution (10 mg HCCA (α-cyano-4-hydroxycinnamic acid) matrix (Bruker Daltonics, Germany) to 1 ml of a solution comprising: 500 µl acetonitrile, 475 µl UHQ water and 25 µl trifluoroacetic acid). 1 µl of the sample-matrix mixture was pipetted onto a MALDI ground steel target plate (Bruker Daltonics) and left to dry on air. MALDI-TOF MS. Mass spectra were acquired using an ultrafleXtreme™ series MALDI-TOF MS (Bruker Daltonics), equipped with a 1 kHz smartbeam-II™ nitrogen laser (λ = 337 nm). Spectra were recorded using the linear positive mode for masses in the range of 2 kDa to 20 kDa. The parameter settings were as follows: ion source 1, 25 kV; ion source 2, 23.85 kV; lens, 6.5 kV; pulse ion extraction time, 160 ns. A low-mass gate of 800 Da was used. Insulin, ubiquitin, cytochrome C and myoglobin were used as external calibrants, enabling a mass accuracy of ±0.5 parts per 1000. Laser power and the number of laser shots per sample were varied by the operator for optimal performance. Data analysis. Mass spectra were processed prior to visual inspection using FlexAnalysis 3.3 software (Bruker Daltonics). This included smoothing, baseline subtraction and intensity normalisation. MALDI BioTyper software 2.0 (Bruker Daltonics) was used to generate pseudo gel view representations of each spectrum. The mass spectra and corresponding pseudo gel images for each bacterial strain were then visually inspected and compared. Preparation of monocytes and S. wadsworthensis (whole cell) stimulation studies Whole venous blood was collected from six adult healthy individuals who were not on any medications. The blood was diluted 1:1 in RPMI 1640 (Sigma-Aldrich), layered onto Histopaque-1077 (Sigma-Aldrich), and centrifuged at 800×g for 30 min. Peripheral blood mononuclear cells were removed from the interface and monocytes were prepared as published earlier [17]. The effect of S. wadsworthensis whole cell preparations was assessed by stimulating the monocytes for up to 24 hours. Seven S. wadsworthensis strains (4 strains obtained from healthy controls and 3 strains from IBD patients) were used in this study along with an E. coli (S1041) as a positive control. Unstimulated monocytes were used as a negative control. Cytometric bead array In order to assess whether the whole cell preparations induced an inflammatory response, levels of TNF-α in culture supernatant were measured at the different time points using a human inflammatory cytokine cytometric bead array (CBA;BD Biosciences). Samples were analyzed on a multifluorescence BD FACSCalibur™ flow cytometer using BD CellQuest™software and BD™ cytometric bead array software. Standard curves were generated using FCAP Array Software™. Sample results were then calculated by comparing sample CBA results to the respective standard curve. All CBA work was performed in duplicate and cytokine levels induced by stimulation were calculated by subtracting unstimulated cytokine levels from each stimulated value and expressing TNF-α levels as a fold-change relative to TNF-α levels derived from E. coli stimulations. Statistical Analysis Statistical analysis was performed using the Pearson Chi Squared, 2-tailed test or the Fisher's exact test wherever appropriate, utilizing Graph Pad software (San Diego, CA).

Discussion The role of S. wadsworthensis in human gastrointestinal diseases has been documented in the past but its specific involvement in inflammatory bowel disease has never been firmly established [8], [9]. The fortuitous isolation of this unusual bacterial strain from mucosal biopsies of patients with IBD prompted a series of experiments that have been outlined in this paper and has culminated in detailed characterization of this putative pathogen. Despite this detailed microbial analysis no specific differences have been identified between cases and controls. However, this negative result does not preclude the importance of successful isolation of this fastidious organism from colonic biopsy samples by adhering to a strict microaerophilic culture environment and stringent growth conditions. Our study provides an important proof of concept that will enable future studies to satisfy Koch's postulates to tie in other luminal microaerophilic bacteria as causative factors in IBD. This is the first report of the appearances of this bacterial species under scanning electron microscopy (SEM). As opposed to previous descriptions, the bacteria are pleomorphic, with strains existing in predominantly two morphological forms, long rods and coccobacilli. Filamentous and helical forms were also documented. The bacteria appeared in clusters on Gram stain and this was further corroborated with the appearance of a lattice type network on SEM. The functional role of this lattice formation is unclear, but given the high prevalence of this organism that we have demonstrated in the human colonic mucosa, further investigation of the interaction of S. wadsworthensis in the complex luminal microbiota may be of interest. This lattice structural network may paradoxically harbour either commensal bacteria or pathogenic bacterial strains in close juxtaposition to the epithelial surface with diametrically opposite consequences. All S. wadsworthensis strains were negative for urease, catalase and oxidase, consistent with the initial description by Wexler et al [8]. The authors had described S. wadsworthensis as asaccharolytic, and certainly none of the sugar assimilation tests from the API kit were found to be positive [8]. As S. wadsworthensis is phenotypically closely related to the Campylobacter genus, the API Campy kit, specifically for the identification of Campylobacter species was trialed. This yielded more promising results. All of the S. wadsworthensis strains tested were positive for the EST, ArgA and AspA tests, meaning they possessed the enzymes esterase, L-arginine arylamidase and L-aspartate arylamidase respectively. Esterases are often required to breakdown dietary residue and are mostly derived from the intestinal microbiota, with E. coli, Bifidobacterium and Lactobacillus species all known to be sources [19]. Colonic esterases have been shown to release powerful antioxidants, present in cereal bran, from complex ester-linked structures which could otherwise not be absorbed [20]. It is possible that the esterases produced by S. wadsworthensis have a similar role. This study utilized a newly developed PCR to detect the presence of S. wadsworthensis in a subset of adult patients with UC and adult control patients. The rates of detection was similar in both these groups (83.8% vs. 86.1%, p = 0.64), suggesting that this bacterial strain is probably a commensal. The only other study that has looked at the prevalence of this pathogen in patients with gastrointestinal disease was by Engberg et al. who used PCR and DNA sequencing of the 16S rRNA gene to characterize bacterial populations in fecal samples [21]. They detected S. wadsworthensis in only 7 out of 1483 (0.47%) patients with GI disorders, and only 1 out of 107 (0.93%) healthy individuals. This wide disparity in prevalence between this study and our cohort of patients and controls suggests that S. wadsworthensis is closely adherent to the mucosal lining and is more likely to be identified from biopsy samples as opposed to feces. Additionally, this newly designed PCR for this bacterial strain was validated using the type strain of S. wadsworthensis as well as several related bacterial strains and will form a useful tool in future studies. Despite similar rates of detection of S. wadsworthensis from patients with IBD and controls, our clinical isolates gave us an excellent opportunity to further characterize this organism. Several bacterial species have been noted to have distinctly different phenotypic and genotypic profiles when isolated from patients with clinical disease as opposed to controls. This has been recently documented in Campylobacter concisus and mucosa associated E. coli wherein distinct genomospecies have been documented with varying pathogenic potential [22], [23]. Sequence analysis of strains identified during the study from cases and controls did not cluster in distinct groups confirming that this phenomenon does not extend to S. wadsworthensis. Proteomic analysis of S. wadsworthensis strains utilizing MALDI-TOF MS is being reported for the first time in this paper. MALDI-TOF MS has recently emerged as an alternative to phenotypic and genotypic methods for the fast and reliable identification of microorganisms down to the species level [16], [24], [25], [26]. It can be used to classify closely related bacteria, which may be indistinguishable by conventional methods [24]. It was therefore employed to investigate the diversity of the S. wadsworthensis protein profiles. A characteristic pattern was clearly demonstrated on the analysis of S. wadsworthensis strains, with a dominant peak at approximately 9400Da. Although no major differences were detected between the strains, MALDI-TOF MS proved to be a highly sensitive and reproducible method for the characterization of the bacterial mass spectra. Very small mass of sample were required, which was ideal for analyzing the slow and modest growing S. wadsworthensis. This finding will provide a useful reference for future studies and an alternative to genotypic methods like 16S rRNA gene sequencing. The in-vitro cytokine analysis after monocyte challenge failed to show any distinct differences between strains isolated from patients with IBD and controls. This last finding essentially closes the loop in the series of studies looking at the possible role of S. wadsworthensis in IBD. Our experiments have conclusively shown that the prevalence of this bacterium is similar in IBD patients and controls and that there is no phenotypic, genotypic, proteomic or pathogenic characteristic to distinguish bacteria isolated from these two groups of patients. It is quite likely that S. wadsworthensis is a commensal and a harmless bystander amidst the inflammatory cascade that is typical of inflammatory bowel disease.

Acknowledgments We would like to thank all adult gastroenterologists (Aberdeen Royal Infirmary) and paediatric gastroenterologists (Royal Aberdeen Children's Hospital and Royal Hospital for Sick Children, Glasgow) for their assistance in recruiting patients to this study. We would also like to thank the patients, children and families who gave their time and consent to participate in this research. We gratefully acknowledge the technical assistance of Mr Ian Davidson for help with MALDI-TOF MS and Mr Kevin MacKenzie for expertise in SEM. We also thank Bruker Daltonics for providing the MALDI BioTyper software.

Author Contributions Conceived and designed the experiments: IM RH EME-O GLH. Performed the experiments: IM RH CEN YAA SHB CP DAS. Analyzed the data: IM RH JMT RKR GLH. Contributed reagents/materials/analysis tools: JMT RKR EME-O. Wrote the paper: IM RH GLH.