Spatial distribution of Escherichia coli in the mouse large intestine inferred from rRNA in situ hybridization

Abstract

Fluorescent oligonucleotide probes targeting rRNA were used to develop an in situ hybridization technique by which the spatial distribution of Escherichia coli in the large intestines of streptomycin-treated mice was determined. Single E. coli cells were identified in thin frozen sections from the large intestines by the use of a probe specific for E. coli 23S rRNA. Furthermore, the total bacterial population was visualized with an rRNA probe targeting the domain Bacteria. By this technique, all E. coli cells were seen embedded in the mucosal material overlying the epithelial cells of the large intestine, and no direct attachment to the epithelium was observed.

Long‐term evolution of the natural isolate of Escherichia coli 536 in the mouse gut colonized after maternal transmission reveals convergence in the constitutive expression of the lactose operon

“…The combination of the massive initial intake of E. coli during inoculation and the early streptomycin-driven dysbiosis (Garner et al, 2009) perturbed the mice gut environment making it less "natural" than hoped. However, the rapid initial decrease in E. coli density previously observed (Poulsen et al, 1994) and the rapid microbiota resilience after the end of streptomycin administration (Stecher et al, 2007) suggest that these initial perturbations are short-lived.…”

Colonization resistance and microbial ecophysiology: using gnotobiotic mouse models and single-cell technology to explore the intestinal jungle

“…mucus-specific adhesins, metabolization of mucin components). It has been demonstrated using FISH that growth of both commensal and pathogenic E. coli strains mainly takes place in the mucus layer of the large intestine (Poulsen et al, 1994;Moller et al, 2003). Other enteropathogens, such as Salmonella enterica serovar Typhimurium (S. Typhimurium), Citrobacter rodentium, and V. cholerae were shown to grow in the mucus layer (Freter et al, 1981;Stecher et al, 2004Bergstrom et al, 2010).…”

Developing a quantitative approach for determining the in vitro prebiotic potential of dietary oligosaccharides

“…These were commercially synthesised and labelled with the fluorescent dye Cy3 (provided by Eurogentec UK Ltd.). The probes used were Bif164 [6], Bac303 [7], Lab158 [8], Erec482 and His150 [9], Ec1531 [10] and Srb687 [11], specific for bifidobacteria, bacteroides, Lactobacillus/Enterococcus spp., Clostridium coccoides-Eubacterium rectale group, Clostridium histolyticum group, E. coli and Desulfovibrio spp., respectively. For total bacterial counts, the nucleic acid stain 4,6-diamidino-2-phenylindole (DAPI) was used.…”

“…The cells were then centrifuged at 1500g for 5 min, washed twice with phosphate-buffered saline (PBS; 0.1 M, pH 7.0), resuspended in a mixture of PBS/99% ethanol (1:1 w/v) and stored at )20°C for at least 1 h. For the Lab 158 probe, samples were further resuspended in the enzyme mixture containing agents (25 mM Tris-HCl, 10 mM EDTA, 585 mM sucrose, 5 mM CaCl 2 , 0.3 mg/ml À1 sodiumtaurocholate, 2 mg/ml À1 lysozyme, 0.1 mg/ml À1 lipase) that increase cell permeability and incubated for 1 h at 37°C. The cells were then washed in PBS, resuspended in 100% methanol and stored at )20°C for at least 1 h. The cell suspension was then added to the hybridisation mixture and left overnight to hybridise at the appropriate temperature for each probe [6][7][8][9][10][11]. Hybridised mixture was vacuum filtered using a 0.2 m Isopore membrane filter (Millipore Corporation, Herts, UK).…”