Colonization with Murine pks+ Escherichia coli under Non-Inflammatory Conditions

Summary

This protocol describes the necessary steps for colonization of mice with pks+ Escherichia coli under non-inflammatory conditions, as well as non-invasive methods for validation of colonization and assessment of pks+ E. coli expansion in feces.

Abstract

pks+ Escherichia coli is increasingly implicated in the development of colorectal cancer (CRC), particularly among a rapidly expanding subset of younger patients with early-onset CRC. pks+ E. coli strains produce the genotoxin colibactin, known to cause double-strand DNA breaks and mutations in cell proliferation and differentiation pathways associated with CRC. This article details a method for pks+ E. coli colonization in mice under non-inflammatory conditions, from preparation of bacterial inoculum through to validation of colonization. Following overnight incubation, pks+ E. coli strain E. coli NC101 is administered to antibiotic-conditioned mice via oral gavage. Colonization is assessed in stool by quantifying the number of lactose-fermenting gram-negative colony forming units (CFUs) and by quantitative PCR amplification. Using this protocol, it was shown that, following the successful colonization of C57BL/6 mice with E. coli NC101, supplementation with the dietary fiber inulin promoted the growth of pks+ E. coli, reflected by increased recovery of CFUs, higher abundance of the Enterobacteriaceae family, and increased levels of the clbP gene. In summary, this model allows for the study of preventative and therapeutic treatments targeting a key pathobiont involved in CRC development.

Introduction

The gut microbiota is known to play a significant role in host health and disease, with gut microbial dysbiosis, or an overgrowth of certain potentially pathogenic bacteria (pathobionts), identified as a hallmark of several disease states, including cardiovascular disease, inflammatory bowel diseases (IBD), and cancer¹. pks+ Escherichia coli is a pathobiont that is enriched in colonic tissue samples of patients with IBD² and colorectal cancer (CRC)³ compared to healthy individuals. The pro-tumorigenic activity of pks+ E. coli is linked to the expression of the polyketide synthase (pks) gene cluster and the production of the genotoxin colibactin⁴. Colibactin translocates to the nucleus of colon epithelial cells, leading to the formation of double-strand DNA breaks⁵ and mutations in key CRC-associated pathways⁶. pks+ E. coli colonization has been shown to contribute to tumor initiation⁷ and tumor progression³ in preclinical models of colitis-associated CRC (CAC). Recently, colibactin-induced mutagenesis has been associated with early-onset CRC^8,9. The incidence of early-onset CRC, occurring in those younger than 50 years of age, has been increasing in recent decades, with the potential to reverse the current overall decrease in CRC rates⁹.

The role of pks+ E. coli in CRC development, as well as in other microbiota-mediated aspects of host health, continues to be elucidated. In addition, several factors have been shown to influence pks gene expression, including inflammation¹⁰, luminal iron concentrations¹¹, and dietary factors^12,13, opening the door for microbiota-targeting interventions. A method is described here for pks+ E. coli intestinal colonization via oral gavage in mice using a murine strain of pks+ E. coli strain NC101 (E. coli NC101). Other models have assessed the effects of E. coli NC101 on inflammation and carcinogenesis in germ-free (GF) mice mono-colonized by oral gavage and rectal swabbing³, in genetically-susceptible (IL-10^-/-) models of colitis^3,14, and in chemically induced models of colon inflammation and gut barrier disruption¹⁵. However, cost-effective colonization with pks+ E. coli can also be obtained under non-inflammatory conditions in specific-pathogen-free (SPF) mice pre-treated with antibiotics to create a niche for colonization and expansion^14,16,17. Various methods for the detection and quantification of pks+ E. coli in mouse feces are also presented, including quantitative PCR (qPCR) amplification of the clbP gene, qPCR assessment of abundance of the family Enterobacteriaceae, and the recovery of colony-forming units (CFUs) on MacConkey agar media specific for gram-negative lactose-fermenting bacteria. These methods may be used interchangeably or in conjunction to confirm the presence of pks+ E. coli and the production of colibactin for validation purposes or to assess differences in pks+ E. coli expansion in response to microbiota-targeting interventions.

Here, this protocol is used to assess the effect of the dietary fiber inulin on pks+ E. coli colonization and expansion by qPCR and recovery of CFUs. Inulin is a prebiotic that has been shown to promote the growth of beneficial Bifidobacterium and Lactobacillus species in the gut, as well as increase the production of short-chain fatty acids, which play an important role in intestinal homeostasis¹⁸. However, inulin has also been shown to promote the growth of pks+ E. coli and colibactin production, leading to increased tumorigenesis in a DSS-treated adenomatous polyposis coli (Apc)^Min/+ CRC model. This experiment evaluates pks+ E. coli expansion in response to dietary inulin in a mouse model without inflammation or an impaired gut barrier.

Protocol

All procedures were performed according to Canadian Council of Animal Care guidelines following approval by the Institutional Animal Care Committee of the Centre de recherche du Centre hospitalier de l'Université de Montréal (CRCHUM). Seven-week-old mice were obtained from a commercial source and were acclimatized for one week prior to the start of the experiment. A schematic representation of this method is presented in Figure 1. The details of the animals, reagents, and equipment used in this study are listed in the Table of Materials.

Figure 1: Schematic of pks+ E.coli preparation and colonization. Day (D)-4: Four days prior to gavage, start mice on antibiotics. D-1: 24h prior to gavage, remove antibiotics. Prepare pks+ E. coli culture for overnight incubation. D0: Prepare the pks+ E. coli bacterial inoculum. Administer the pks+ E. coli or a vehicle control (saline) to mice via oral gavage as soon as possible after preparation of the inoculum. D7+ Collect feces from mice for the detection and quantification of pks+ E. coli colonization. Please click here to view a larger version of this figure.

1. Antibiotic treatment

1. Dilute ampicillin, colistin, and streptomycin in sterile drinking water to a final concentration of 1 mg/mL for ampicillin, 1 mg/mL for colistin, and 5 mg/mL for streptomycin.
2. Allow ad libitum access to antibiotics in drinking water for 3 days.
3. Remove antibiotics and restore access to sterile water for a 24-h washout period before the gavage with E. coli NC101 inoculum.
   NOTE: Antibiotic solution should be prepared fresh for each experiment. Adequate intake of the antibiotic solution should be monitored. If necessary, a sweetener may be added to the water.

2. Preparation of E. coli NC101 inoculum (up to n = 12 mice)

NOTE: Steps 2.1-2.8 should be performed using aseptic technique.

1. Prepare and autoclave Lysogeny Broth (LB).
2. Streak E. coli NC101 from glycerol stock onto an LB agar plate using a sterile inoculating loop. Incubate overnight at 37 °C.
3. Inoculate 5mL of sterile LB broth in a culture tube with a single colony from the LB agar plate using an inoculating loop. Incubate overnight at 37 °C in a benchtop incubation shaker at 150 rpm.
4. Centrifuge the culture tube at 1500 x g for 5 min at room temperature.
5. Remove and discard the supernatant, avoiding the bacterial pellet, which will appear as an off-white deposit at the base of the tube.
6. Resuspend the bacterial pellet in 5 mL of 0.9% sterile saline.
7. Wash by repeating steps 4 to 6.
8. Measure the optical density at 600 nm (OD₆₀₀) using a cell density meter and adjust the OD₆₀₀ to 0.625 (~1 x 10⁸ CFUs/mouse) using sterile saline.
   NOTE: To ensure no bacterial contamination of the broth in step 3, incubate a separate tube of 2-3 mL of sterile LB broth overnight. The broth should remain clear after overnight incubation, indicating no bacterial contamination. If the starting OD₆₀₀ is less than 0.625, repeat steps 4 to 6, using a smaller volume to resuspend the pellet. To ensure accurate dosage of E. coli NC101, serial dilutions of the gavage liquid should be plated and cultured overnight. The number of CFUs for each dilution may be used to estimate the initial CFU amount in the bacterial suspension.

3. Oral gavage of E. coli NC101

1. Gently agitate the bacterial inoculum to ensure an equal distribution of bacteria.
2. Fit the gavage needle onto a 1 mL syringe and fill with 0.2 mL of bacterial inoculum, removing any air bubbles.
3. Remove the mouse from the cage and gently but firmly scruff the mouse by grasping the skin from just behind the ears to the shoulder blades, using the thumb pad and the side of the index finger. Secure the tail with the middle or ring finger from the same hand.
4. Hold the mouse in an upright position and insert the gavage needle on the side of the mouth. Glide the end of the gavage needle along the roof of the mouth and into the esophagus and stomach, gently pushing back the mouse's head to extend the neck. If any resistance is felt, remove the gavage needle. Re-scruff the mouse if necessary before trying again.
5. Slowly inject the bacterial inoculum (0.2 mL) and withdraw the needle, following the same angle as the insertion.
6. Return the mouse to the cage and proceed with the remaining mice.
   NOTE: The bacterial inoculum should be administered as soon as possible after preparation. Control mice should receive 0.2 mL of 0.9% sterile saline (vehicle). The appropriate length, gauge, and ball diameter of the gavage needle should be determined based on the average body weight of the mice in each experiment. Check the breathing pattern and activity of the mouse after 5 min and before leaving the animal facility. If laboured breathing/respiratory distress is observed, euthanize the mouse following an approved protocol. Oral gavage should only be performed by trained research staff. Improper execution of oral gavage can lead to serious complications or death due to oesophageal perforation or injection of the gavage liquid into the lungs. When performed by experienced research staff, no mortality is expected with this procedure. Mice used for this experiment should be housed under SPF conditions. In order to prevent contamination between cages, individually ventilated cages should be used, and mice should be manipulated in a biological safety cabinet. Before opening each cage, the hands and the outside of the cage lid should be wiped with an appropriate disinfectant (e.g., accelerated hydrogen peroxide). The PPE requirements for manipulating mice used in this protocol include a gown (disposable or reusable) and gloves. Additional PPE may be required by the host animal facility. All manipulations involving control mice should be performed first.

4. Collection of fecal samples

1. Collect a fecal pellet from each mouse in a 1.5 mL microcentrifuge tube by scruffing the mouse as described in step 3.3. The mouse should defecate immediately. Alternatively, place the mouse in a clean empty cage and wait up to 30 min for defection to occur.
2. Snap-freeze the sample by placing the tube in liquid nitrogen. Store the sample at -80 °C until further analysis.
   NOTE: Detection of clbA or quantification of clbP in stool by real-time PCR should be performed prior to the start of the experiment to verify that mice are not colonized with pks+ E. coli. In order to maintain colonization beyond one week, re-occurring bacterial gavage without the pre-administration of antibiotics (steps 2.1-3.6) may be necessary, depending on the experimental conditions. Quantification of E. coli NC101 in stool by CFU recovery or real-time PCR should be performed at least weekly for the duration of the experiment when implementing this protocol for the first time to optimize dosing frequency.

5. Fecal bacteria DNA extraction

1. Extract bacterial DNA from collected stool samples using a DNA extraction kit following the manufacturer's instructions.
2. Measure the DNA concentration using a spectrophotometer and adjust the DNA concentration to 5 ng/µL.

6. Confirmation of colonization by detection of clbA and E. coli 16S genes

1. Perform simultaneous amplification of the clbA and E. coli 16S ribosomal RNA (rRNA) genes (Supplementary Table 1) by PCR using an appropriate master mix and instrument for PCR following the manufacturer's instructions.
2. Visualize clbA and E. coli 16S genes in E. coli NC101 colonized mice by agarose gel electrophoresis as previously described¹⁹. Non-colonized mice should lack the clbA band.

7. Quantification of E. coli NC101 by CFU recovery

NOTE: Perform steps 7.1-7.7 using aseptic technique.

1. Weigh an empty 1.5 mL microcentrifuge tube.
2. Add roughly 20 mg of stool to the 1.5 mL microcentrifuge tube, and record the weight of the tube with the added sample.
3. Add 200 µL of 0.9% sterile saline to each tube.
4. Wait 10-15 min for stools to soften at room temperature and vortex each tube for 5 min at maximum speed to yield a homogeneous suspension. Small particles of undigested food material will be visible.
5. Transfer 100 µL of each suspension into a MacConkey agar plate and evenly spread the homogenate using an L-shape spreader.
6. Incubate the plates overnight at 37 °C.
7. Count the number of pink colonies and normalize by the weight of each fecal pellet and the volume plated.
   NOTE: MacConkey agar selects for gram-negative bacteria. Additionally, bacteria that can ferment lactose (such as E. coli) form pink colonies, while those that cannot form colourless or yellow colonies. Only pink colonies should be counted for the purposes of this protocol. If the colony number is too elevated to count, serial dilutions of the initial homogenate may be performed using 0.9% sterile saline. Take into account the dilution factor when calculating the number of colonies per sample.
   Pellet weight = weight of microcentrifuge tube with the pellet sample - weight of empty microcentrifuge tube.

8. Quantification of the clbP gene

1. Perform amplification of the clbP and universal 16S rRNA genes (Supplementary Table 1) by quantitative PCR using an appropriate master mix and instrument for real-time PCR following the manufacturer's instructions.

9. Quantification of Enterobacteriaceae

1. Perform amplification of the Enterobacteriaceae and universal 16S RNA genes (supplementary) by quantitative PCR using an appropriate master mix and instrument for real-time PCR.

Results

The amplification and visualisation of the clbA and E. coli 16S genes in feces at the endpoint by PCR and gel electrophoresis showed that, while both genes were visible in E. coli NC101-colonized mice, only the E. coli 16S gene was visible in control mice, confirming that E. coli NC101 was not present (Figure 2B).

As expected¹⁵, E. coli NC101 colonized mice that received a diet supplemen...

Discussion

The pro-carcinogenic effects of colibactin-producing pks+ E. coli have recently been associated with both the rise in early-onset CRC⁹ and the adoption of a "Western-style" diet¹², factors that will continue to shape CRC trends for the coming decades. Most models of pks+ E. coli colonization are shown in the context of colitis-associated cancer, which do not allow for the characterization of pks+ E. coli under non-inflammatory conditions. ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.9% sterile saline	Baxter	JF7634
10% Inulin custom diet	Teklad	TD.240286	Representative results
50 ppm FeS04 (No-fiber) custom diet	Teklad	TD.120515	Representative results
8-12 week old female B6 mice	Charles River Laboratories	C57BL/6	Representative results
Ampicillin sodium salt	Wisent Inc	400-110 XG
Benchtop centrifuge	Fisher scientific	75004381
Cell density meter	Biochrom	80-2116-30
Culture tube, 13mL	Sarstedt	62.515.006
EcNC101	N/A	N/A	Gifted by Dr. Christian Jobin, University of North Carolina at Chapel Hill
Gavage needle, 38mm x 22 G	Harvard Apparatus Canada	34-024	No longer available - A potential alternative is available at Instech Labs (FTP-22-38)
Incubating orbital shaker	VWR	980153
Inoculating loop	Fisher scientific	22-363-595
L-shape spreader	Fisher scientific	14-665-230
Lysogeny broth	Wisent Inc	800-060 LG	Add 35g of LB powder 1L of water. Autoclave before using.
Lysogeny broth agar	Wisent Inc	800-011 LG	Add 35g of LB powder 1L of water. Autoclave before using.
MacConkey agar	Becton Dickinson	211387	Add 50g of MacConkey powder to 1L water. Autoclave before using.
Microbial DNA extraction kit	Qiagen	47016
Microbiological incubator	Fisher scientific	150152633
Microcentrifuge tube, 1.5mL	UltiDent Scientific	73-M150-C
PCR Rotor -Gene strip tubes	UltiDent Scientific	73-V100-RG
Petri dish	Sarstedt	82.1472.001
Real-time PCR SYBR Green Master Mix	ThermoFisher	A25742
Real-time PCR thermal cycler	Corbett Research	RG-3000A
Screw cap tube, 15mL	Sarstedt	62.554.502
Sodium colistimethate	Fresenius Kabi	C309306
Streptomycin sulfate salt	Millipore Sigma	S9137
Tuberculin slip tube, 1mL	Becton Dickinson	309659
Vortex	Fisher scientific	12-812