In Vivo Colonization Test of Two CRISPR-Engineered Escherichiacoli in Mice

doi:10.12300/j.issn.1674-5817.2022.088

Abstract

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 2×10 ¹⁰ 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.

Key words: CRISPR engineered Escherichia coli, Intestinal colonization capacity, Colonization efficiency, ICR mouse

CLC Number:

Q95-33

Chenyin REN, Siqi GAO, Hao LI, Biao TANG, Hua YANG, Yuehuan LIU. In Vivo Colonization Test of Two CRISPR-Engineered Escherichiacoli in Mice[J]. Laboratory Animal and Comparative Medicine, 2022, 42(6): 541-550.

Figures/Tables 6

Figure 1 Structure of the E. coli I CRISPR-Cas system (A), plasmid profile of pGLO-J23100-GFP in Nissle1917 (B), and plasmid profile of pGLO-araC-GFP in BW25113 (C)Note：J23100， constitutive promoter； RBS， ribosome binding site； GFP， green fluorescent protein expression gene （fluorescent reporter system element）； Promoter， gene promoter； Amp R， ampicillin resistance selection marker gene； araC， ara operon regulatory protein.

Figure 2 Fluorescence in stock solution and dilution solution of E. coli engineered bacteria as well the diet and 10% glucose waterNote: A, Stock solution of engineered bacteria (100×); B-C, Dilution solution of engineered bacteria (B,×100; C, ×1 000); D-F, Green, red and blue fluorescence in diet (×200); G, Fluorescence in 10% glucose water (×200)

Figure 3 Amp + plate detection of the colonization of two E. coli strains in mouse tissuesNote: A-C, Nissle1917 at 24, 48, and 72 h after gavage, respectively；D-F, BW25113 at 24, 48, and 72 h after gavage, respectively; 1, 2, and 3 are engineered E. coli experimental groups; 4 and 5 are the PBS control groups; from the left to the right, and from the top to the bottom on the same plate: mesenteric lymph nodes, stomach, ileocecal and colon tissues.

Figure 4 Fluorescence microscopic detection of adhesion of 2 strains of engineered E. coli in the intestinal tissue of mice at 24 h, 48 h, and 72 h after gavage (×200)Note: A, Nissle1917 in the ileocecal area at 24 h; B, Nissle1917 in the colon at 24 h; C, BW25113 in the ileocecal area at 24 h; D, BW25113 in the colon at 24 h; E, Nissle1917 in the ileocecal area at 48 h; F, Nissle1917 in the colon at 48 h; G, BW25113 in the ileocecal area at 48 h; H, BW25113 in the colon at 48 h; I, Nissle1917 in the ileocecal area at 72 h; J, Nissle1917 in the colon at 72 h; K, BW25113 in the ileocecal area at 72 h; L, BW25113 in the colon at 72 h. Arrows show attached glowing bacteria.

Figure 5 Electrophoresis of PCR products of Nissle1917 and BW25113Note： M, DNA marker (DL2 000); P, Positive control (Nissle1917/BW25113); N, Negative control (PBS). Lanes 1–6: Gavage 24 h (lanes 1, 3 and 5 show ileocecal, lanes 2, 4 and 6 show colon); Lane 7-12: Gavage 48 h (lanes 7, 9 and 11 show ileocecal, lanes 8, 10 and 12 show colon); Lane 13-18: Gavage 72 h (lanes 13, 15 and 17 show ileocecal, lanes 14, 16 and 18 show colon).

Figure 6 Comparison of the colonization efficiency of Nissle1917 and BW25113 (gavage 24–72 h) in different tissues of mice

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