A Simple In Vitro Gut Model for Studying the Interaction between Escherichia coli and the Intestinal Commensal Microbiota in Cecal Mucus

Abstract

A novel in vitro gut model was developed to better understand the interactions between Escherichia coli and the mouse cecal mucus commensal microbiota. The gut model is simple and inexpensive while providing an environment that largely replicates the nonadherent mucus layer of the mouse cecum. 16S rRNA gene profiling of the cecal microbial communities of streptomycin-treated mice colonized with E. coli MG1655 or E. coli Nissle 1917 and the gut model confirmed that the gut model properly reflected the community structure of the mouse intestine. Furthermore, the results from the in vitro gut model mimic the results of published in vivo competitive colonization experiments. The gut model is initiated by the colonization of streptomycin-treated mice, and then the community is serially transferred in microcentrifuge tubes in an anaerobic environment generated in anaerobe jars. The nutritional makeup of the cecum is simulated in the gut model by using a medium consisting of porcine mucin, mouse cecal mucus, HEPES-Hanks buffer (pH 7.2), Cleland's reagent, and agarose. Agarose was found to be essential for maintaining the stability of the microbial community in the gut model. The outcome of competitions between E. coli strains in the in vitro gut model is readily explained by the "restaurant hypothesis" of intestinal colonization. This simple model system potentially can be used to more fully understand how different members of the microbiota interact physically and metabolically during the colonization of the intestinal mucus layer. IMPORTANCE Both commensal and pathogenic strains of Escherichia coli appear to colonize the mammalian intestine by interacting physically and metabolically with other members of the microbiota in the mucus layer that overlays the cecal and colonic epithelium. However, the use of animal models and the complexity of the mammalian gut make it difficult to isolate experimental variables that might dictate the interactions between E. coli and other members of the microbiota, such as those that are critical for successful colonization. Here, we describe a simple and relatively inexpensive in vitro gut model that largely mimics in vivo conditions and therefore can facilitate the manipulation of experimental variables for studying the interactions of E. coli with the intestinal microbiota.

Generation of 13C-Labeled MUC5AC Mucin Oligosaccharides for Stable Isotope Probing of Host-Associated Microbial Communities

“…While heavy labeled MUC5AC is relevant to the lung, cervix, eye, GI tract, and middle ear, we acknowledge that Calu-3-derived mucins have their limitations. Most notably, Calu-3 cells are an adenocarcinoma cell line, which are known to express altered glycosylation patterns and compromised biosynthetic regulation relative to normal tissues, and may not reflect mucin glycan composition that varies with body site or disease state. ,, However, given that crude preparations of MUC5AC derived from porcine gastric mucosa or bovine maxillary glands are the commonly used and currently accepted commercially available substrates for in vitro studies of mucin–microbe interactions, ,− Calu-3-derived mucins described here can be regarded as a rational model for SIP-based studies of mucosal-associated microbiota. Future studies will be aimed at generalizing the approach to other immortalized cell lines or even primary cell culture, which will likely enable the isolation of other secreted and tethered mucins.…”

Local Delivery of Streptomycin in Microcontainers Facilitates Colonization of Streptomycin-Resistant Escherichia coli in the Rat Colon

“…Here, we have demonstrated that microcontainers can be utilized for local delivery of antibiotics in the gut environment to selectively enhance the proliferation of an introduced resistant gut bacterium in the cecum and colon. We chose streptomycin as the model antibiotic, as this bactericidal drug is conventionally added to the drinking water at 5g/L to allow in vivo colonic establishment of orally introduced Gram-negative bacteria, such as E. coli ( 14 – 19 ). However, we found that this concentration of streptomycin also affected the small intestinal bacterial community and allowed E. coli engraftment also in this compartment.…”