Depletion of Murine Intestinal Microbiota: Effects on Gut Mucosa and Epithelial Gene Expression

Abstract

BackgroundInappropriate cross talk between mammals and their gut microbiota may trigger intestinal inflammation and drive extra-intestinal immune-mediated diseases. Epithelial cells constitute the interface between gut microbiota and host tissue, and may regulate host responses to commensal enteric bacteria. Gnotobiotic animals represent a powerful approach to study bacterial-host interaction but are not readily accessible to the wide scientific community. We aimed at refining a protocol that in a robust manner would deplete the cultivable intestinal microbiota of conventionally raised mice and that would prove to have significant biologic validity.Methodology/Principal FindingsPreviously published protocols for depleting mice of their intestinal microbiota by administering broad-spectrum antibiotics in drinking water were difficult to reproduce. We show that twice daily delivery of antibiotics by gavage depleted mice of their cultivable fecal microbiota and reduced the fecal bacterial DNA load by 400 fold while ensuring the animals' health. Mice subjected to the protocol for 17 days displayed enlarged ceca, reduced Peyer's patches and small spleens. Antibiotic treatment significantly reduced the expression of antimicrobial factors to a level similar to that of germ-free mice and altered the expression of 517 genes in total in the colonic epithelium. Genes involved in cell cycle were significantly altered concomitant with reduced epithelial proliferative activity in situ assessed by Ki-67 expression, suggesting that commensal microbiota drives cellular proliferation in colonic epithelium.ConclusionWe present a robust protocol for depleting conventionally raised mice of their cultivatable intestinal microbiota with antibiotics by gavage and show that the biological effect of this depletion phenocopies physiological characteristics of germ-free mice.

Exploring host–microbiota interactions in animal models and humans

“…By following communities before, during, and after treatment with antibiotics, significant insight can be gained into not only basic questions of microbial ecology and microbiome function (Maurice et al 2013), but also how antibiotics-among the most widely used pharmaceuticals-impact susceptibility to infectious microbes and disease susceptibility. The temporal changes in luminal and mucosal microbial populations and their coincident impact on intestinal lymphoid populations, epithelial gene expression, and peripheral lymphoid tissues have been examined in response to antibiotics (vancomycin, metronidazole, neomycin, and ampicillin 6 amphotericin-B) provided in the drinking water or by gavage (Hill et al 2010;Reikvam et al 2011). Antibiotic perturbations of the mouse microbiota have shed insight into how vancomycin-resistant enterococcus (VRE), an organism that causes significant morbidity and mortality, can come to dominate the gut microbiome and set the stage for VRE bacteremias, which are increasingly seen in hospitalized patients (Ubeda et al 2010).…”

Microbiota and Gut Stem Cells Cross-Talks: A New View of Epithelial Homeostasis

“…GF rats have a slight hyperplasia of their intestinal villi, which is associated with a comparatively short cell cycle time. Similarly, in the colon of GF or antibiotic-treated mice, the cell proliferation rate is reduced and their crypts contain fewer cells than those of conventional mice [5,6]. Several other alterations have been shown in the gut of GF animals, such as reduction of the villous capillary network, a reduction in digestive enzymes activity, the presence of smaller Peyer's patches, and impairment in the gut peristaltic activity [1].…”

Investigating a holobiont: Microbiota perturbations and transkingdom networks

“…[29][30][31][32] Furthermore, a few studies compared both germfree and antibiotic-treated animals and detected concordant results. 31,33 However, concordant results are not always observed between these 2 models. For example, in an animal model of common variable immunodeficiency (CVID) associated enteropathy (in B lymphocyte deficient mice), we found that derivation of B cell knockout (BcKO) and control mice as germfree abolished differences in the host phenotype between the 2 genotypes observed in conventional mice ( Fig.…”