Transcriptional activity of the dominant gut mucosal microbiota in chronic inflammatory bowel disease patients

Abstract

Dysbiosis of the gut mucosa-associated microbiota (MAM) plays a pivotal role in the pathogenesis of chronic inflammatory bowel diseases (IBD). To date, dysbiosis only describes the altered composition of the different bacterial populations, but little is known about transcriptional activity, metabolism and the 'live' status of the MAM. In this study we investigated the transcriptional activity of the dominant intestinal bacterial populations in patients with IBD. Colonic mucosal biopsies from patients with active Crohn's disease (CD; n510), active ulcerative colitis (UC; n510) and healthy individuals (HI; n510) were compared by 16S rRNA gene and rRNA profiles using clone libraries with more than 1700 sequenced clones. Bacterial richness was significantly lower in clone libraries based on rRNA compared to those based on the rRNA genes in the CD group (3.01 vs 3.91) and the UC group (3.61 vs 4.15), but showed no difference in HI (3.81 vs 3.85). The qualitative composition of rRNA and rRNA gene clone libraries was significantly different, with the phylum Bacteroidetes being the most active (P,0.01) compared to other populations in all clinical groups. In contrast, Actinobacteria and Firmicutes were inactive in the CD group, while Escherichia sp. were both abundant and active in the CD and UC groups. Most of the phylotypes showing the highest activity index ratios represented less than 1 % of the microbiota. Our findings indicate that specific bacterial populations are activated in IBD patients, while other groups are in an inactive or 'dormant' state. The transcriptional activity points to a more functional role of the intestinal mucosal microbiota and may lead to the identification of therapeutic targets in the active modulation of microbial factors. INTRODUCTIONChronic inflammatory bowel diseases (IBD) are characterized by dysbiosis of the intestinal microbiota (Ott et al., 2004;Tamboli et al., 2004;Manichanh et al., 2006). Dysbiosis is discussed as a major factor in disease pathogenesis along with genetic mutations and a dysregulation of the immune response to bacterial antigens (Schreiber et al., 2005). Loss of natural intestinal diversity and a shift of bacterial composition towards a more deleterious profile might reflect the net effect of environmental influences over the past few decades leading to the dramatic increase of the incidence of IBD in the industrialized world (Ott et al., 2004(Ott et al., , 2008aManichanh et al., 2006). To date, dysbiosis only describes the altered composition of the different bacterial populations, but little is known about transcriptional activity, metabolism and 'live' status of the mucosa-associated microbiota (MAM), which may have more relevance for the host, e.g. by direct contact with the gut epithelium, secretion of pro-inflammatory cytokines, quorum-sensing molecules or other microbe-associated molecular patterns.A maximum of 10-30 % of the total microbes of the complex bacterial ecosystem within the gut can be cultivated using classical microbiological approaches (McFa...

Dynamic changes of the luminal and mucosa-associated gut microbiota during and after antibiotic therapy with paromomycin

“…Active mucosa-associated B. dorei prevailed in patients with ulcerative colitis (UC) compared to healthy individuals and has been associated with active celiac disease as well as collagenous colitis. 18,25,26 OTU 3137 (Clostridium hiranonis) is an indicator for luminal microbiota at the DNA level and for mucosa at the RNA level (further discussed in the last section). C. hiranonis strains dehydroxylate primary bile acids to form secondary bile acids, which are important for an adequate lipid metabolism.…”

“…Moreover, it is of importance to take the active microbiota into account, as only they contribute to metabolic turnover and were shown to differ from present microbiota in healthy and diseased people. 18,19 To our knowledge, there is currently no study exploring both luminal and mucosal microbiota simultaneously at the abundance and activity level.…”

Uso da biblioteca genômica RNAr 16S como ferramenta para o estudo da microbiota fecal humana

“…Este fenômeno é conhecido como "resistência à colonização", "interferência microbiana" ou "efeito barreira" (GARRETT et al, 2010;VRIEZE et al, 2010;DAVID et al, 2005 As análises moleculares de comunidades complexas foram primeiramente empregadas em estudos ambientais analisando de forma abrangente como os ecossistemas respondem às perturbações ambientais (Rondon, et al, 2000;BRITSCHGI e GIOVANNONI,1991;GIL et al, 2006 Análises filogenéticas baseadas em PCR e construção de biblioteca genômica de RNAr 16S têm sido utilizadas para caracterizar a microbiota de fezes humana (HAYASHI et al, 2003;HAYASHI et al, 2005). Os diferentes estudos da microbiota intestinal utilizando o método de amplificação do gene RNAr 16S disponíveis na literatura mostram uma grande diversidade e variedade dos componentes bacterianos desta microbiota (LI et al, 2012;DURBAN et al, 2011;REHMAN et al, 2010;WALKER et al, 2011;SHEN et al, 2010;LIN et al, 2013).…”

“…Os trabalhos mostrando a microbiota intestinal de seres humanos revelam a utilização do iniciador 27F HAYASHI et al, 2002;HAYASHI et al, 2005, LI et al, 2012REHMAN et al, 2010;WALKER et al, 2011;MAI et al, 2013;TOTTEY et al, 2013). No estudo da análise molecular da microbiota fecal de idosos descrito por HAYASHI e colaboradores (2003) A construção da biblioteca genômica RNAr 16S tem sido amplamente utilizada para caracterizar a microbiota intestinal humana ao longo das últimas duas décadas, mesmo sendo uma metodologia de custo significativo e de intenso trabalho (HAYASHI et al, 2005;STRAUSBERG et al, 2008;LIN et al, 2013;DURBÁN, et al, 2011;REHMAN et al, 2010;WALKER et al, 2011;SHEN et al, 2010).…”

“…No estudo da análise molecular da microbiota fecal de idosos descrito por HAYASHI e colaboradores (2003) A construção da biblioteca genômica RNAr 16S tem sido amplamente utilizada para caracterizar a microbiota intestinal humana ao longo das últimas duas décadas, mesmo sendo uma metodologia de custo significativo e de intenso trabalho (HAYASHI et al, 2005;STRAUSBERG et al, 2008;LIN et al, 2013;DURBÁN, et al, 2011;REHMAN et al, 2010;WALKER et al, 2011;SHEN et al, 2010). Embora a construção da biblioteca de RNAr 16S possa resultar numa avaliação menos sensível da diversidade bacteriana, revela diferenças significativas entre os membros da microbiota intestinal e permite a caracterização dos organismos mais abundantes, que podem ser melhor adaptadas para o seu nicho (ZOETENDAL et al, 2004;Li et al, 2010; RICUCCI e SIQUEIRA, 2010).…”