NEWS & VIEWS GUT MICROBIOTA

Optimal sampling of the intestinal microbiota for research R. Balfour Sartor Refers to Lavelle, A. et al. Spatial variation of the colonic microbiota in patients with ulcerative colitis and control volunteers. Gut http://dx.doi.org/10.1136/gutjnl-2014-307873

This commentary outlines our expanding understanding of resident bacterial compartmentalization along the longitudinal and vertical intestinal axes and suggests optimal approaches to sampling the microbiota. Although readily available and noninvasive, faecal microbiota are not fully representative of mucosally associated bacterial communities, which might uniquely interact with epithelial and immune cells. Humans coexist with a biologically and metabolically active intestinal microbiota that promote mucosal homeostasis by stimulating protective regulatory immune and epithelial responses in healthy hosts, but drives pathogenic immune-mediated intestinal inflammation and tissue injury in genetically susceptible individuals. 1 Dysbiosis—an altered balance of protective and aggressive resident bacteria—is associated with multiple inflammatory and metabolic conditions, including Crohn’s disease, ulcerative colitis, obesity, metabolic syndrome, IBS and alcoholic liver disease, with evidence that these abnormal microbial community structures are both a cause and a consequence of the disease processes. The majority of studies examining resident enteric microbial profiles have relied on readily accessible faecal samples obtained on a single occasion, despite mounting evidence that faecal analyses might not properly represent mucosally associated bacterial populations.2,3 This disparity is an important concern because many investigators believe that the mucosally associated microbiota might affect epithelial and mucosal function to a greater degree than luminal bacteria. Now, Lavelle et al.4 address the important questions of whether spatial variation of the resident bacterial communities occurs in different regions of the colons of healthy volunteers as controls and of patients with active ulcerative colitis, and in luminal and mucosal compartments. In an intensive analysis of a small number of control individuals undergoing

colonoscopy with normal intestines (n = 4) and patients with ulcerative colitis undergoing a colectomy for refractory active disease (n = 5), these investigators carefully collected multiple samples from four colonic regions ranging from the caecum to the rectum. 4 Samples were collected by brushing the luminal microbiota, by mucosal biopsies and laser capture microdissection of the mucus gel layer closely adherent to the colonic mucosa from snapfrozen biopsy samples. Comprehensive deep sequencing of the bacterial 16S ribosomal RNA gene yielded >31,000 reads per sample and the data were analysed by modern bioinformatic techniques. These carefully performed studies showed the expected large interindividual variation in the luminal and mucosally associated microbiota, but demonstrated very little difference between the bacterial community structures of the caecum, transverse colon, descending colon and rectum in either the luminal or mucosal compartments of an individual. However, within each region, the microbial profiles varied between the lumen and mucus gel layer, with mucosal biopsy sample results intermediate; a relatively small subset of bacterial families were responsible for the observed disparity (Figure 1). These observations provide a guide to explore the function of the bacterial families differentially located in the lumen versus the surface mucus gel. Surprisingly, these researchers found increased bacterial community diversity and increased concentrations of Clostridiaceae in the inflamed samples,4

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in contrast to most other observations.1,2 This finding could perhaps be explained by the lack of a bowel preparation in the patients with ulcerative colitis undergoing resection compared with controls and the fact that samples were collected by colonoscopic biopsies in controls, but from surgically resected tissues from patients with r­efractory ulcerative colitis.

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…faecal bacterial community structures do not fully replicate mucosally associated profiles

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These results have important implications for optimal sampling of the gut microbiota in clinical and translational research studies. Despite the convenience, low cost and noninvasive advantages of faecal sampling, faecal bacterial community structures do not fully replicate mucosally associated profiles.2–4 However, it seems that the global bacterial patterns of rectal biopsy samples might sufficiently mirror those of the more proximal colon.2–4 Studies in mice suggest that ileal luminal bacteria are strikingly different between luminal colonic and faecal profiles,5 but findings from studies of the ileal versus colonic mucosal bacterial community structure in humans have not yet reached a consensus.2,6 Lavelle et al.4 unfortunately did not investigate ileal luminal contents or mucosal specimens, but presented novel information of bacterial community structure in the tightly mucosally adherent colonic mucus gel layer isolated by laser capture microdissection. Microbial composition of this difficult to access mucus compartment can also be studied by in situ staining (fluo­ rescence in situ hybridization or immuno­ histochemistry), but these approaches require directed probes of selected bacterial families or species and are not amenable to nontargeted deep-sequencing techniques. 7,8 Fortunately, Lavelle et al. 4 demonstrated that bacterial profiles contained within standard forceps mucosal biopsy samples adequately represent bacterial populations within the technically difficult to access dissected gel layer so that standard mucosal sampling is sufficient to assess mucosal microbiota. ADVANCE ONLINE PUBLICATION  |  1

NEWS & VIEWS Technical studies are needed to determine whether post-collection washing of biopsy samples would eliminate loosely adherent luminal bacteria not representative of the bacterial populations within the mucus gel. Biopsies have the advantage of capturing not only mucosally adherent microbiota, but also invasive microbial species, including true pathogens, functionally abnormal resident bacteria (such as adherent–invasive Escherichia coli, also termed AIEC) and species translocating across the compromised mucosal barrier of Crohn’s disease.8 Techniques need to be developed to easily and reliably separate the mucus gel from biopsy samples to identify mucosally invasive and translocating bacterial species of particular importance to disease pathogenesis. In situ staining can provide semiquantitative information on invasive bacteria, but are restricted to t­argeted analysis, as mentioned earlier.7,8 This new study 4 and previous work provide clear evidence that both luminal and mucosal sampling are necessary to adequately characterize the complex micro­biota of the distal colon. We assume that faecal populations closely represent distal colonic

luminal microbiota, but this concept needs to be carefully documented and confirmed for humans as subtle differences can occur in mice.9 Longitudinal analyses of intestinal microbiota changes with disease progression and response to therapy are important and necessary requisites for optimal clinical application of microbial-directed therapies.10 Clinically applicable serial sampling necessarily requires noninvasive methods, with faecal collection being the most practical approach, perhaps with a single or at most two biopsies to extend results to the mucosal compartment. A key unmet need is to determine the relative importance of luminal and mucosally associated microbiota in driving normal homeostatic and disease-inducing and/or disease-perpetuating activities. A testable hypothesis is that the larger mass of luminal bacteria in conjunction with dietary and sloughed mucosal substrates produce the majority of bacterial metabolites, whereas mucosally adherent or invasive bacteria more directly induce epithelial and immune responses (Figure 1). In addition, we need to identify and validate optimal sampling methods and the spatial variation of

Healthy

sIgA β-defensin

Lumen

Dietary components

Intestinal epithelial cell

Bacteria IL-10 TGF-β

Dysbiosis

Immune cells Metabolic products (SCFAs)

Lumen ■ Bacteroidaceae Mucus layer ■ Lachnospiraceae* ■ Coriobacteriaceae ■ Ruminococcaceae ■ Family X1 Incertea Sedis ■ Planctomycetaceae*

Ulcer Paneth cell

Bacterial translocation TLR ligands

UNC Multidisciplinary IBD Center, University of North Carolina at Chapel Hill, Room 7309A Molecular Biomolecular Research Building, 111 Mason Farm Road, CB# 7032, Chapel Hill, NC 27599‑7032, USA. [email protected] doi:10.1038/nrgastro.2015.46 Published online 24 March 2015 Competing interests R.B.S. is on a standing advisory panel (North American Probiotic Council) for Danone and Yakult, and receives grant support from GlaxoSmithKline and Salix for microbial-related studies. He has been on advisory boards for Vertex, Salix and GlaxoSmithKline related to microbiome studies. 1.

Inflamed

Outer mucus layer Inner mucus layer

intestinal fungi and viruses that might be as important to mucosal homeostasis and intestinal disease pathogenesis as the more thoroughly studied bacterial communities. Optimizing sampling methods and timing of longitudinal collection of intestinal microbiota will help achieve the enormous therapeutic potential of selectively normalizing the abnormal microbial community in individuals with a microbial-driven disease such as IBD and help guide personalized approaches to restoring normal complex bacterial communities to prevent recurrent Clostridium difficile infection.

IFN-γ IL-17 TNF IL-1β IL-6 IL-8 IL-12

Lumen Mucus layer ■ Porphyromonadaceae* ■ Peptostreptococcaceae ■ Bacteroidaceae* ■ Bifidobacteriaceae ■ Veillonellaceae ■ Ruminococcaceae ■ Enterobacteriaceae ■ Coriobacteriaceae

Figure 1 | Resident bacteria in the luminal and mucosal compartments in inflamed & and healthy Nature Reviews | Gastroenterology Hepatology conditions. Complex resident bacteria communities exist in both the lumen and the mucosally attached mucus gel, which is composed of a loose outer layer colonized by a defined population of resident bacteria and a relatively sterile inner mucus layer where sIgA and secreted defensins are concentrated, forming an antimicrobial shield. These bacterial communities differ in healthy and inflamed (Crohn’s disease and ulcerative colitis) conditions, with some bacterial families unique to particular states (marked by an asterisk). Abbreviations: SCFA, short-chain fatty acid; sIgA, secretory IgA; TGF‑β, transforming growth factor β.

2  |  ADVANCE ONLINE PUBLICATION

Sartor, R. B. The intestinal microbiota in inflammatory bowel diseases. Nestle Nutr. Inst. Workshop Ser. 79, 29–39 (2014). 2. Gevers, D. et al. The treatment-naive microbiome in new-onset Crohn’s disease. Cell Host Microbe 15, 382–392 (2014). 3. Stearns, J. C. et al. Bacterial biogeography of the human digestive tract. Sci. Rep. 1, 170 (2011). 4. Lavelle, A. et al. Spatial variation of the colonic microbiota in patients with ulcerative colitis and control volunteers. Gut http://dx.doi.org/ 10.1136/gutjnl-2014-307873. 5. Gu, S. et al. Bacterial community mapping of the mouse gastrointestinal tract. PLoS ONE 8, e74957 (2013). 6. Zhang, Z. et al. Spatial heterogeneity and co‑occurrence patterns of human mucosalassociated intestinal microbiota. ISME J. 8, 881–893 (2014). 7. Swidsinski, A. et al. Spatial organization and composition of the mucosal flora in patients with inflammatory bowel disease. J. Clin. Microbiol. 43, 3380–3389 (2005). 8. Baumgart, M. et al. Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn’s disease involving the ileum. ISME J. 1, 403–418 (2007). 9. Gulati, A. S. et al. Mouse background strain profoundly influences paneth cell function and intestinal microbial composition. PLoS ONE 7, e32403 (2012). 10. Hansen, J. J. & Sartor, R. B. Therapeutic manipulation of the microbiome in IBD: current results and future approaches. Curr. Treat. Options Gastroenterol. 13, 105–120 (2015).

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Gut microbiota: Optimal sampling of the intestinal microbiota for research.

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