Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Summary

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Abstract

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Introduction

EHEC O157:H7 is a pathogen that causes diarrhea¹, HS², HUS³, and even acute renal failure⁴ through contaminated water or food. EHEC is a pathogenic enterobacterium and colonizes to the gastrointestinal tract of humans¹. When EHEC first adhere to host intestinal epithelium, they inject the colonization factors into host cells through the type III secretion system (T3SS) that functions as a molecular syringe inducing an attaching and effacing (A/E) lesion subsequently to enforce adhesion (colonization)⁵. These genes involved in ....

Protocol

Caution: EHEC O157:H7 is a biosafety level 2 (BSL-2) pathogen according to the Centers for Disease Control and Prevention (CDC) biosafety instruction (https://www.cdc.gov/). Therefore, all experimental procedures involving EHEC must be performed in a BSL-2 facility. Wear lab coats and gloves while performing the experiment. Work in a certified biosafety cabinet (BSC). Disinfect the experimental bench before and after the experimental procedure with 70% ethanol. All instruments or equipment that contact (or potentially contact) EHEC should be disinfected with 70% ethanol or bleach. Contaminated (or potentially contaminated) wastes should be sealed and autoclaved carefu....

Results

We administered bioluminescence-labeled EHEC (~ 10⁹ bacterial cells) to 6-week old female C57BL/6 mice by oral gavage. After oral inoculation of EHEC to mice within 1 h, the animals were examined for bioluminescent signal by the in vivo imaging system as shown in Figure 7. The results showed a strong bioluminescent signal in gavage mice with bioluminescence-labeled EHEC. We examined the signals on 2 days post infection. As shown in

Discussion

It has been reported that EHEC transformed with luciferase plasmid has been utilized to examine its localization in hosts or gene expression in vivo^8,11,12. The murine model demonstrated here has also been reported to detect the EHEC colonized timing and localization in murine host⁸. Nevertheless, we provide the detail protocol of how to administer EHEC inoculation to mice intragastrically and ho.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Shaker incubator	YIH DER	LM-570R	bacteria incubation
Orbital shaking incubator	FIRSTEK	S300	bacteria incubation
pBSL180			source of nptII gene
pAKlux2			source of luxCDABE operon
T&A Cloning Kit	Yeastern Biotech	FYC001-20P	use for TA cloning
Nsi I	NEB	R0127S	use for plasmid cloning
Sca I	NEB	R0122S	use for plasmid cloning
Spe I-HF	NEB	R0133S	use for plasmid cloning
Sma I	NEB	R0141S	use for plasmid cloning
T4 ligase	NEB	M0202S	use for plasmid cloning
Ex Taq	TaKaRa	RR001A	use for PCR amplification
10X Ex Taq Buffer	TaKaRa	RR001A	use for PCR amplification
dNTP Mixture	TaKaRa	RR001A	use for PCR amplification
PCR machine	applied Biosystem	2720 thermal cycler	for PCR amplification
Glycerol	SIGMA	G5516-1L	use for bacteria stocking solution
NaCl	Sigma	31434-5KG-R	chemical for making LB medium, 10 g/L
Tryptone	CONDA pronadisa	Cat 1612.00	chemical for making LB medium, 10 g/L
Yeast Extract powder	Affymetrix	23547-1 KG	chemical for making LB medium, 5 g/L
Agar	CONDA pronadisa	Cat 1802.00	chemical for making LB agar
kanamycin	Sigma	K4000-5G	antibiotics, use for seleciton
streptomycin	Sigma	S6501-100G	antibiotics, eliminate the microbiota in mice
EDL933 competent cell	Homemade		method is on supplemental document
Electroporator	MicroPulser		for electroporation
Electroporation Cuvettes	Gene Pulser/MicroPulser	1652086	for electroporation
High-speed centrifuge	Beckman Coulter	Avanti, J-26S XP	use for centrifuging bacteria
Fixed-Angle Rotor	Beckman Coulter	JA25.5	use for centrifuging bacteria
Fixed-Angle Rotor	Beckman Coulter	JLA10.5	use for centrifuging bacteria
centrifuge bottles	Beckman Coulter	REF357003	use for centrifuging bacteria
centrifuge bottles	Thermo Fisher scientific	3141-0500	use for centrifuging bacteria
eppendorf biophotometer plus	eppendorf	AG 22331 hamburg	for measuring the OD600 value of bacteria
C57BL/6 mice	Laboratory Animal Center of NCKU
lab coat, gloves			for personnel protection
isoflurane	Panion & BF Biotech Inc.	G-8669	for mice anesthesia, pharmaceutical grade
1ml syringe			use for oral gavage of mice
Reusable 22 G ball-tipped feeding needle		φ0.9 mm X L 50 mm	use for oral gavage of mice
surgical  scissors			use for mice experiment
Xenogen IVIS 200 imaging system	Perkin Elmer	IVIS spectrum	use for bioluminescent image capture
Living Image Software	Perkin Elmer	version 4.1	use for quantifying the image data