Name: Colonization with Murine pks+ Escherichia coli under Non-Inflammatory Conditions (Video) | JoVE Journal
Uploaded: 2026-03-10T14:10:00.000+00:00
Duration: 7 min 46 s
Description: Learn a protocol for colonizing mice with pks+ Escherichia coli under non-inflammatory conditions to study host-microbiota interactions in colorectal cancer research. This method enables controlled bacterial colonization without inflammation confounding.

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5 minutes

This work was funded by a grant from the Canadian Institutes of Health Research [CIHR, grant CSV-198232]. We thank the animal facility of the CRCHUM. Figures were created using BioRender.com.

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.
<img alt="E. coli experiment timeline, diagram of antibiotic treatment, culture preparation, oral gavage procedure." class="xfigimg" src="/files/ftp_upload/70204/70204fig1.jpg" data-altupdatedat="1781582408000" data-alt="Figure 1"> 
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. <a href="https://www.jove.com/files/ftp_upload/70204/70204fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Antibiotic treatment
<ol>
	<li>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.</li>
	<li>Allow ad libitum access to antibiotics in drinking water for 3 days.</li>
	<li>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.</li>
</ol>
2. Preparation of E. coli NC101 inoculum (up to n = 12 mice)
NOTE: Steps 2.1-2.8 should be performed using aseptic technique.
<ol>
	<li>Prepare and autoclave Lysogeny Broth (LB).</li>
	<li>Streak E. coli NC101 from glycerol stock onto an LB agar plate using a sterile inoculating loop. Incubate overnight at 37 °C.</li>
	<li>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.</li>
	<li>Centrifuge the culture tube at 1500 x g for 5 min at room temperature.</li>
	<li>Remove and discard the supernatant, avoiding the bacterial pellet, which will appear as an off-white deposit at the base of the tube.</li>
	<li>Resuspend the bacterial pellet in 5 mL of 0.9% sterile saline.</li>
	<li>Wash by repeating steps 4 to 6.</li>
	<li>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.</li>
</ol>
3. Oral gavage of E. coli NC101
<ol>
	<li>Gently agitate the bacterial inoculum to ensure an equal distribution of bacteria.</li>
	<li>Fit the gavage needle onto a 1 mL syringe and fill with 0.2 mL of bacterial inoculum, removing any air bubbles.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Slowly inject the bacterial inoculum (0.2 mL) and withdraw the needle, following the same angle as the insertion.</li>
	<li>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.</li>
</ol>
4. Collection of fecal samples
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
5. Fecal bacteria DNA extraction
<ol>
	<li>Extract bacterial DNA from collected stool samples using a DNA extraction kit following the manufacturer's instructions.</li>
	<li>Measure the DNA concentration using a spectrophotometer and adjust the DNA concentration to 5 ng/µL.</li>
</ol>
6. Confirmation of colonization by detection of clbA and E. coli 16S genes
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
7. Quantification of E. coli NC101 by CFU recovery
NOTE: Perform steps 7.1-7.7 using aseptic technique.
<ol>
	<li>Weigh an empty 1.5 mL microcentrifuge tube.</li>
	<li>Add roughly 20 mg of stool to the 1.5 mL microcentrifuge tube, and record the weight of the tube with the added sample.</li>
	<li>Add 200 µL of 0.9% sterile saline to each tube.</li>
	<li>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.</li>
	<li>Transfer 100 µL of each suspension into a MacConkey agar plate and evenly spread the homogenate using an L-shape spreader.</li>
	<li>Incubate the plates overnight at 37 °C.</li>
	<li>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.</li>
</ol>
8. Quantification of the clbP gene
<ol>
	<li>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.</li>
</ol>
9. Quantification of Enterobacteriaceae
<ol>
	<li>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.</li>
</ol>

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The authors have no conflicts of interest to declare.

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 &#34;Western-style&#34; 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. ...

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 CRC8,9. 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 factors12,13, 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 colitis3,14, 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 expansion14,16,17. 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+&#160;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+&#160;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.

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

colonization with murine pks+ escherichia coli under non-inflammatory conditions

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

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+&#160;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.

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...

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.

pks+ Escherichia coli is increasingly implicated in the development of colorectal cancer (CRC), particularly among a rapidly expanding subset of younger ...

This protocol describing PKS positive E.coli colonization and quantification in a mice allows for the study of host microbiota interactions in evolving the key pathobionts implicated in colorectal cancer development. In comparison to other methods that utilize genetically or chemically induced models of colon inflammation, this protocol describes colonization under non-inflammatory condition. To begin add ampicillin, colistin, and streptomycin to a container.And then pour sterile drinking water to dilute the antibiotics. Provide the antibiotic containing drinking water to the eight to 12 week old female C57 BL6 mouse for three days. Then remove the antibiotic containing drinking water and restore access to sterile drinking water for a 24 hour washout period before the gavage.Using a sterile inoculating loop, pick a single colony of Escherichia coli NC101 from the agar plate and inoculate five milliliters of sterile lysogeny broth. Incubate the culture overnight at 37 degrees Celsius in a benchtop incubation shaker at 150 revolutions per minute. Then centrifuge the culture tube at 1500 G four five minutes at room temperature.Remove and discard the supernatant while avoiding disturbance of the bacterial pellet. The bacterial pellet appears as an off-white deposit at the base of the tube. Resuspend the bacterial pellet in five milliliters of 0.9%sterile saline.Repeat the centrifugation step and wash the pellet again in 0.9%sterile saline. After the second wash, measure the optical density of the suspension at 600 nanometers using a cell density meter. Adjust the optical density at 600 nanometers to 0.625, corresponding to approximately one times 10 to the power of eight colony forming units per mouse using sterile saline.Select an appropriate gavage needle based on the size of the animal. Gently agitate the bacterial inoculum to ensure equal distribution of the bacteria. Fill the syringe with 0.2 milliliters of the bacterial inoculum and remove any air bubbles.Remove the mouse from the cage. Scruff the mouse by grasping the skin 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 of the same hand.Hold the mouse in an upright position. Insert the gavage needle into the side of the mouth. Glide the tip of the gavage needle along the roof of the mouth into the esophagus and stomach while gently pushing the mouse head back to extend the neck.Slowly inject 0.2 milliliters of the bacterial inoculum and withdraw the gavage needle following the same angle as insertion. Monitor the mouse five minutes after gavage for signs of respiratory distress and discomfort. Return the mouse to the cage.And place the animal on a diet supplemented with inulin or a no fiber control diet. Collect a fecal pellet from each mouse in a 1.5 milliliter micro centrifuge tube by scruffing the mouse as previously demonstrated. Alternatively, place the mouse in a clean, empty cage and wait up to 30 minutes for defecation to occur.Snap freeze the sample by placing the micro centrifuge tube in liquid nitrogen. If required, freeze the sample at minus 80 degrees Celsius until further analysis. Next, weigh an empty 1.5 milliliter micro centrifuge tube.Add approximately 20 milligrams of stool to the 1.5 milliliter micro centrifuge tube and record the weight. Add 200 microliters of 0.9%sterile saline to the tube and incubate at room temperature to soften the stool. After 10 to 15 minutes, vortex the tube for five minutes at maximum speed to produce a homogeneous suspension.Small particles of undigested food material may remain visible. Transfer 100 microliters of each suspension onto a MacConkey agar plate and evenly spread the homogenate using an L-shaped spreader. Incubate the plates overnight at 37 degrees Celsius.On the following day, count the number of pink colonies on each plate. Normalize the colon accounts by the weight of each fecal pellet and the plated volume. Extract genomic DNA from the fecal samples using a DNA extraction kit.Quantify the CLB genes and enterobacteriaceae abundance using quantitative PCR with gene specific and family specific primers. Endpoint, PCR and gel electrophoresis detected both Colibactin A and E.coli 16S genes in feces from E.coli NC101 colonized mice. In control mice, only the E.coli 16S gene was detected, supporting the absence of PKS plus E.coli colonization.After one week, the E.coli NC101 colonized mice that received the inulin supplemented diet showed greater colony forming unit recovery from the fecal homogenates cultured on MacConkey agar than the mice that received the no fiber control diet. At the same time point, the inulin fed mice also showed increased Colibactin P gene levels. And a higher enterobacteriaceae abundance than the no fiber control mice.This protocol describes several methods for the validation of PKS positive E.coli colonization and the quantification of PKS positive E.coli growth by bacterial culturing and PCR amplification. This protocol can be combined with one of several colorectal cancer models, allowing for the assessment of PKS positive E.coli associated mutagenesis in colon tissue. This protocol can be used to study microbiota targeting methods to limit PKS positive E.coli expansion, and mutagenic potential in healthy hosts.

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Immunology and Infection

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Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

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Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

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Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

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Modeling Ascending Vaginal Infection, Preterm Birth, and Neonatal Morbidity in Mice

Learn a protocol for colonizing mice with pks+ Escherichia coli under non-inflammatory conditions to study host-microbiota interactions in colorectal cancer research. This method enables controlled bacterial colonization without inflammation confounding.

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

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