Microbiome Alterations in Different Phases of Celiac Disease
Stephen M. Lagana M.D.1, Ian S. Cohn2, Mara R. Rubinstein2, Benjamin Lebwohl M.D., MS3, Peter H.R. Green M.D.3, Yiping W. Han Ph.D.2, Govind Bhagat M.D.1
1Department of Pathology and Cell Biology; 2Department of Microbiology & Immunology; 3Department of Medicine, Celiac Disease Center, Columbia University Medical Center, NYPH, College of Physicians and Surgeons, New York, NY, United States.
The proximal small intestine is not considered to have a large microbial burden and until recently there were only limited data regarding the microbial composition of this region. Starting in the mid 2000’s, however, a variety of methodologies e.g. electron microscopy and targeted PCR were used to determine commensal bacteria at this site and also identify alterations in patients with celiac disease (CD). The results varied based on age, diet, environment and antibiotic use. Recent studies using high throughput sequencing approaches have shown an enrichment of Bifidobacterium, Firmicutes and Prevotella sp. in the healthy state and an overrepresentation of E. coli, Fusobacterium, Neisseria and Haemophilus sp. In CD patients with active inflammation.
The purpose of this study was to compare the duodenal microbiome of adult CD patients in different disease phases; newly diagnosed or active CD (ACD, n=19), those on a gluten free diet (GFD, n=14), patients with disease refractory to a gluten free diet, with a normal IEL phenotype and polyclonal products being detected on PCR analysis for TCR-beta gene rearrangement (RCD1, n=24) and age and sex matched “normal” controls (n=15).
DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) duodenal biopsies using the Qiamp FFPE kit (Qiagen, Germantown, USA) according to standard methods and subjected to next generation sequencing (NGS). Briefly, the 16S rRNA gene V4 variable region PCR primers with barcode on the forward primer were used in a 30 cycle PCR. After amplification and checking the quality of the PCR products on a 2% agarose gel, multiple samples were pooled together in equal proportions based on their molecular weight and DNA concentrations. Pooled samples were purified using calibrated Ampure XP beads. The pooled and purified PCR product was used to prepare a DNA library by following Illumina TruSeq DNA library preparation protocol. Sequencing was performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq (2X300 bp, average of 20K reads) following the manufacturer’s guidelines (Illumina, San Diego, USA). The Q25 sequence data were processed using a proprietary bioinformatics pipeline (MR DNA, Shallowater, TX, USA). Sequences were de-noised, operational taxonomic units (OTUs) were generated and chimeras removed. The OTUs were defined by clustering at 3% divergence (97% similarity). Final OTUs were classified taxonomically using BLASTn against a curated database derived from GreenGenes, RDPII and NCBI (www.ncbi.nlm.nih.gov, DeSantis et al 2006, http://rdp.cme.msu.edu) and compiled into each taxonomic level as both, “counts” and “percentage” files.
Mean bacterial burden in different disease states. There is a higher bacterial burden in inflamed states vs. controls (ACD and RCD1 vs. Control p=.05; ACD and RCD vs. Control and GFD p=.09). Microbial composition also differs. Bold indicates significant differences among phyla. Note low actinobacteria levels (commensal organisms) in RCD1.
The most abundantly identified genera in ACD vs. GFD (matched pairs n=4). The only significant difference is the lower levels of Corynebacterium sp. in ACD compared to GFD.
The microbiome of RCD1 differs from GFD patients and more closely resembles the composition of ACD patients. This graph shows the most abundant genera in GFD vs. RCD1. A significant difference in Corynebacterium levels is seen; low in RCD1 and high in GFD. Levels of Fusobacterium and Cloacibacterium are higher in RCD1 compared to patients on GFD.
Taking into account all genera (not just the most abundant), 25 show significant difference in levels between GFD and RCD1 samples (all listed on this graph).
Celiac disease patients with active inflammation (ACD, RCD1) show an increased bacterial burden and alterations of the duodenal flora (genus level) than controls, indicating dysbiosis.
Differences in the duodenal microbial composition of seronegative refractory celiac disease type 1 patients compared to those on a gluten-free diet (and similarities to those with active disease) suggests a link between certain bacteria and inflammation; cause or effect awaits further studies
Further analysis, to determine species level differences, and animal studies will provide further insights into the role of microbiome alterations and inflammation in CD.
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