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Higher Prevalence of Bacteroides Fragilis in Crohn's Disease Exacerbations and Strain-Dependent Increase of Epithelial Resistance

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Higher Prevalence of Bacteroides Fragilis in Crohn's Disease Exacerbations and Strain-Dependent Increase of Epithelial Resistance bioRxiv preprint doi: https://doi.org/10.1101/2020.05.16.099358; this version posted May 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Higher prevalence of Bacteroides fragilis in Crohn's disease exacerbations and strain-dependent increase of epithelial resistance Heike E. F. Beckera,b, Casper Jaminc, Liene Bervoetsa, Pan Xub, Marie J. Pierikb Frank R. M. Stassena, Paul H. M. Savelkoula,d, John Pendersa,c*, Daisy M. A. E. Jonkersb* aDepartment of Medical Microbiology, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Centre+, Maastricht, The Netherlands; bDivision of Gastroenterology/Hepatology, Department of Internal Medicine, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Centre+, Maastricht, The Netherlands; cDepartment of Medical Microbiology, Caphri School for Public Health and Primary Care, Maastricht University Medical Centre+, Maastricht, The Netherlands; dDepartment of Medical Microbiology and Infection Control, Amsterdam University Medical Center, location VUMC, Amsterdam, The Netherlands *Authors contributed equally to this work. Corresponding author: Dr. John Penders, Department of Medical Microbiology, Maastricht University Medical Center+, PO 5800, 6202 AZ, Maastricht, The Netherlands/Prof. dr. Daisy M.A.E. Jonkers, Department of Internal Medicine, Division of Gastroenterology-Hepatology, Maastricht University Medical Centre+, PO 5800, 6202 AZ, Maastricht, The Netherlands bioRxiv preprint doi: https://doi.org/10.1101/2020.05.16.099358; this version posted May 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Higher prevalence of Bacteroides fragilis in Crohn's disease exacerbations and strain-dependent increase of epithelial resistance Abstract Bacteroides fragilis has previously been linked to Crohn’s disease (CD) exacerbations, but results are inconsistent and underlying mechanisms unknown. This study investigates the epidemiology of B. fragilis and its virulence factors bft (enterotoxin) and ubiquitin among 181 CD patients and the impact on the intestinal epithelial barrier in vitro. The prevalence of B. fragilis was significantly higher in active (n=69/88, 78.4%) as compared to remissive (n=58/93, 62.4%, p=0.018) CD patients. Moreover, B. fragilis was associated with intestinal strictures. Interestingly, the intestinal barrier function, as examined by transepithelial electrical resistance (TEER) measurements of Caco-2 monolayers, improved when exposed to secretomes of bft-positive (increased TEER ~160%, p<0.001) but not when exposed to bft- negative strains. Whole metagenome sequencing and metabolomics, respectively, identified 19 coding sequences and two metabolites that discriminated TEER-increasing from non- TEER-increasing strains. This study revealed a higher B. fragilis prevalence during exacerbation. Surprisingly, bft- positive secretomes improved epithelial resistance. Keywords: Bacteroides fragilis; Crohn’s disease; barrier function; prevalence; metabolomics; whole genome sequencing; organoids bioRxiv preprint doi: https://doi.org/10.1101/2020.05.16.099358; this version posted May 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Introduction Crohn’s disease (CD) is a chronic inflammatory disease, characterized by patchy inflammation of the intestinal mucosa with or without extra-intestinal manifestations.1 The disease course varies largely among patients with alternating periods of remission and exacerbations. Insufficient control of the recurrent mucosal inflammation results in phenotype progression and complications, such as strictures of fistulas, contributing to a high disease and economic burden.2,3 CD onset is considered to involve genetic predisposition, environmental factors and an adverse immune reaction to the host microbiota.1 However, the factors influencing the occurrence of exacerbations, complications and disease phenotype remain largely unclear. In recent years, microbial dysbiosis gained increasing attention as a factor contributing to exacerbations.1,4 Several studies reported a decreased microbial diversity and altered microbial composition in active CD patients compared to remission.4–6 As a consequence of compositional changes, alterations in overall microbial functionality are conceivable.7 Since the intestinal epithelium limits bacterial attachment by a mucus layer, bacteria often interact with the host via their secretome, consisting of metabolites, proteins and bacterial membrane vesicles (MVs).8–11 On the one hand, secreted glycosidases and mucinases are able to degrade the mucus layer and allow microbes to directly interact with epithelial cells.10,12 On the other hand, bacterial metabolites, such as the short chain fatty acid butyrate, have been reported to promote mucus production and the sealing capacity of the intercellular junctional complex.13,14 In CD, an impaired intestinal epithelial barrier function has increasingly been recognized as a hallmark of exacerbations.1 The impaired barrier function has been associated with alterations in the epithelial junctional complex.15,16 It remains unknown whether the observed altered microbiota composition and functionality during CD exacerbations can contribute to this impaired epithelial barrier. In addition to alterations in microbial diversity, specific microbial taxa were detected in CD exacerbations4,17,18 of which Bacteroides fragilis is a prominent example.4,17,19 Several studies investigated the colonization of B. fragilis during exacerbation and remission in fecal samples and biopsies. Together, the results are inconclusive and based on rather low sample sizes.4,17,19,20 So far, it is still unclear whether the prevalence of B. fragilis differs in different disease stages of CD and how it may affect disease activity. B. fragilis is a Gram-negative commensal of the phylum Bacteroidetes and some strains secrete various virulence factors, such as an eukaryotic-like ubiquitin (Ubb) and B. fragilis toxin (Bft; fragilysin).21,22 It is yet unclear whether these virulence factors contribute to CD exacerbations. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.16.099358; this version posted May 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. To gain more insights into the interaction between the intestinal microbiota and the intestinal barrier in CD, especially during dysbiosis, research essentially needs to focus on microbial functionality, such as reflected by the secretome. In this study, we therefore aim to investigate firstly the prevalence and relative abundance of B. fragilis and virulence-factor positive strains in a large, well-defined cross-sectional CD patient cohort and secondly the impact of the B. fragilis secretome on the intestinal epithelial barrier as first site of interaction and pathophysiological factor in CD exacerbations. We hypothesize that B. fragilis and its virulence factors bft and ubb are involved in exacerbations and by disrupting the intestinal epithelial barrier. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.16.099358; this version posted May 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Results Patient population In total, 181 CD patients, 88 with active disease and 93 in remission, from the IBD South Limburg (IBDSL)23 cohort fulfilling the inclusion criteria were available for the present study. Baseline characteristics were comparable between patient groups, although steroid use was slightly higher among patients with active disease (table 1). Table 1. Baseline characteristics of CD patient cohort. CD remission (n=93) CD exacerbation (n=88) Age (median in years; IQR) 43.0 (32.0-56.5) 45.0 (28.0-57.0) Gender (% female) 63.4 51.1 Calprotectin (median; IQR) 22.0 (14.0-55.5) 476.5 (325.8-703.0) Montreal classification (%) A1; A2; A3 6.5; 67.7; 25.8 10.2; 61.4; 28.4 L1; L2; L3; L4 35.5; 17.2; 47.3; 4.3 23.9; 21.6; 54.5; 11.4 B1; B2; B3; p 59.1; 19.4; 31.2; 17.2 62.5; 25.0; 17.1; 11.4 Surgery 34.4 31.8 IBD medication use (%) Thiopurine 34.4 29.6 Biologicals 58.1 48.9 Steroids 17.2 30.7 Other 13.4 18.2 L1 = Terminal ileum; L2 = Colon; L3 = Ileocolon; A1 < 16 years, A2 = 17-40 years, A3 > 40 years at diagnosis; B1 = without structure formation, non-penetrating; B2 = Stricturing; B3 = Penetrating B. fragilis prevalence and relative abundance Based on the detection of the B. fragilis specific gyrB gene, the prevalence of B. fragilis was significantly higher among CD patients during an exacerbation (78.4 %) when compared to those in remission (62.4 %, p = 0.018, figure 1A). We subsequently determined the prevalence of the B. fragilis virulence factors bft and ubb. Only 11.4 % and 7.5 % (p = 0.376) of the samples from the exacerbation and remission group, respectively, were positive for bft. When considering only samples that were tested positive for B. fragilis, this equals to a proportion of bft-positive samples of 14.5 % and 12.1 % (p = 0.689), respectively. For ubb, the prevalence was 19.3 % and 11.8 % (p = 0.164) in the entire exacerbation and remission group, respectively, and 24.6 % and 19.0 % (p = 0.442), when considering the gyrB positive samples bioRxiv preprint doi: https://doi.org/10.1101/2020.05.16.099358; this version posted May 16, 2020.
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