Shiyan dongwu yu bijiao yixue (Dec 2022)
In Vivo Colonization Test of Two CRISPR-Engineered Escherichia coli in Mice
Abstract
Objective The colonization ability and efficiency of two Escherichia coli (E. coli)-engineered strains, Nissle1917 and BW25113, in the intestine were evaluated in mice, we aimed to screen out strains for subsequent research on clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system-engineered bacteria to eliminate drug-resistant bacteria via high intestinal colonization efficiency. Methods Seventy ICR mice (18–20 g), half male and half female, were randomly divided into 7 treatment groups by gender, with 10 mice in each group (6 for experiment and 4 for control). The experimental group was gavaged with 2×1010 of the engineered strains at a final volume of 200 μL, and the control group was gavaged with an equal volume of PBS. At 1, 3, 6, 12, 24, 48, and 72 hours after gavage, the mesenteric lymph nodes, stomach, ileocecal and colonic tissues, and intestinal contents of the mice were removed. The two E. coli strains were detected using plate inoculation, fluorescence microscopy, and PCR amplification to compare their in vivo colonization ability and efficiency. Results At 1, 3, 6, 12, 24, and 48 hours after gavage, both E. coli strains had colonized in the stomach, ileocecal and colonic tissues as detected using the three methods, and no leakage of E. coli fluid from the lymph nodes was observed; at 72 hours, only Nissle1917 colonized in the ileocecal and colonic tissues, comparing the colonization efficiency of the two E. coli strains, that of Nissle1917 was 100% and that of BW25113 was 0 at 72 hours. Conclusion Nissle1917 has a higher colonization efficiency than BW25113 and can colonize in the mucosal surface of ileocecal and colonic tissues for a long time, suggesting that it can be used as a carrier for the CRISPR system to prevent and control drug resistance gene transmission.
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