Molecular analysis of the gut microbiota of identical twins with Crohn's disease

Abstract

Increasing evidence suggests that a combination of host genetics and the composition of the gut microbiota are important for development of Crohn's disease (CD). Our aim was to study identical twins with CD to determine microbial factors independent of host genetics. Fecal samples were studied from 10 monozygotic twin pairs with CD (discordant n ¼ 6 and concordant n ¼ 4) and 8 healthy twin pairs. DNA was extracted, 16S rRNA genes were PCR amplified and T-RFLP fingerprints generated using general bacterial and Bacteroides group-specific primers. The microbial communities were also profiled based on their percentage G þ C contents. Bacteroides 16S rRNA genes were cloned and sequenced from a subset of the samples. The bacterial diversity in each sample and similarity indices between samples were estimated based on the T-RFLP data using a combination of statistical approaches. Healthy individuals had a significantly higher bacterial diversity compared to individuals with CD. The fecal microbial communities were more similar between healthy twins than between twins with CD, especially when these were discordant for the disease. The microbial community profiles of individuals with ileal CD were significantly different from healthy individuals and those with colonic CD. Also, CD individuals had a lower relative abundance of B. uniformis and higher relative abundances of B. ovatus and B. vulgatus. Our results suggest that genetics and/or environmental exposure during childhood, in part, determine the gut microbial composition. However, CD is associated with dramatic changes in the gut microbiota and this was particularly evident for individuals with ileal CD.

Impact of chicory inclusion in a cereal-based diet on digestibility, organ size and faecal microbiota in growing pigs

“…To ensure appropriate lysis of bacteria, the heating step was supplemented with three cycles of heating at 958C for 5 min followed by snap-freezing in liquid nitrogen. To obtain a profile of the faecal microbiota composition, extracted DNA was analysed with the T-RFLP methodology as previously described (Dicksved et al, 2008). Briefly, bacterial 16S rRNA gene fragments were PCR amplified from each DNA extract with general primers Bact-8F 5 0 -end labelled with 6-carboxyfluorescein and 926r.…”

“…Cloning and sequencing To identify bacteria that corresponded to TRFs of interest, small clone libraries from six pigs were created as previously described (Dicksved et al, 2008). Briefly, 16S rRNA gene fragments of the extracted DNA were amplified using primers Bact-8F and 926r.…”

The Gut Microbiota: Ecology and Function

“…We have performed extensive studies on a Swedish twin cohort that include healthy individuals and individuals with different inflammatory bowel diseases (see more detail later). Our initial T-RFLP data revealed a high degree of similarity in the gut microbiota of identical twins, even when they lived apart for decades (Dicksved et al, 2008).…”

“…In this chapter we primarily discuss CD because of the large number of recent reports that have focused on the correlation of the gut microbiota to this particular disease. Different molecular fingerprinting techniques, including T-RFLP (Dicksved et al, 2008;Willing et al, 2009a), TGGE (Seksik et al, 2005), qPCR (Willing et al, 2009a), and 454 pyrosequencing (Willing et al, unpublished data), have revealed that the gut microbiota is different in individuals with CD compared to healthy individuals. This altered gut microbiota has been referred to as a state of "dysbiosis" (Seksik et al, 2005;Tamboli et al, 2004).…”

Association of Obesity with Serum Leptin, Adiponectin, and Serotonin and Gut Microflora in Beagle Dogs

“…Culture‐independent technique has been used in microbiome studies of dog 19, 20. Culture‐independent technique approach allowed us to bypass the isolation and cultivation of individual species and improved our ability to identify obesity‐associated microbial changes in the gut 21, 22…”