Apr 03, 2025

Public workspaceCitrobacter rodentium Model of Enteropathogenic and Enterohemorrhagic (EPEC, EHEC) Escherichia coli Infection

DOI
dx.doi.org/10.17504/protocols.io.dm6gpdej8gzp/v1
  • 1Department of Microbiology and Immunology, McGill University, Montreal, Canada
Alexandra Kazanova
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DOI: dx.doi.org/10.17504/protocols.io.dm6gpdej8gzp/v1
Protocol Citation: Alexandra Kazanova, samantha.gruenheid 2025. Citrobacter rodentium Model of Enteropathogenic and Enterohemorrhagic (EPEC, EHEC) Escherichia coli Infection. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpdej8gzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 11, 2025
Last Modified: April 03, 2025
Protocol Integer ID: 124537
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000525
Abstract
Purpose: To outline preparation of inoculum and per os Citrobacter rodentium infection in mice.
Guidelines
Brief safety overview:

Citrobacter rodentium is a Gram-negative enteric bacterium and Bio-Safety Level (BSL) 2 pathogen. Containment level 2 practices, safety equipment, and facilities are recommended for work involving infectious or potentially infectious materials, animals, or cultures.

Abbreviations:
CFU: Colony forming Unit
LB: Luria-Bertani
PBS: phosphate-buffered saline
RPM: rotations per minute

Note
N.B. Diet has a large impact on infection, we wean mice onto Teklad 2918 and maintain them on this diet.

ABC
Mouse strainsAgeOutcome
C57BL/64-8 weeksresistant: peak bacterial burden at day 12 p.i. but mice recover
Materials
  • Citrobacter rodentium strain DBS100 (chloramphenicol-resistant) used for CFU experiments
  • 75% ethanol spray bottle
  • Autoclaved 50% glycerol
  • LB broth: Dissolve and mix Amount10 g Bacto tryptone (casein extract), Amount5 g yeast extract, and Amount10 g NaCl in Amount1 L dH2O then autoclave.
AB
Bacto tryptone (casein extract)10 g
Yeast extract5 g
NaCl in 1L dH2O10 g
  • MacConkey agar plates: Dissolve and mix Amount50 g in Amount1 L dH2O, heat with frequent agitation, boil for Duration00:01:00 to completely dissolve powder and autoclave (Temperature121 °C , Duration00:15:00 ) (Difco; REF 212123)
  • MacConkey agar Petri dishes supplemented with chloramphenicol: Use chloramphenicol at final concentration of Amount20 µL (Amount667 µL of Amount30 µL stock in Amount1 L MacConkey)
  • Sterile 1x PBS
  • Autoclaved glass beads for spreading bacteria on Petri dishes
  • Orbital shaker
  • 37°C incubator
  • Sterile bacterial glass culture tubes
  • 5ml push cap tubes (Sarstedt; 55.526.006)
  • Ice bucket
  • Gavage needle (22G)
  • X machine in BSL2
  • Glass beads
  • diam. 1.0mm (sterile)
  • V-bottom 2ml cryotubes (sterile)
  • Analytical balance
  • MagNA lyser

ReagentTube, 5 ml, (LxØ): 75 x 12 mm, PPSarstedtCatalog # 55.526.006
ReagentMacConkey Agar DifcoCatalog #212123











Prepare Master Stock of Citrobacter rodentium (1 time)
Prepare Master Stock of Citrobacter rodentium (1 time)
On day 1, streak Citrobacter rodentium wild-type or chloramphenicol-resistant DBS100 strains from Amount1 mL frozen stocks on LB agar plates (with or without chloramphenicol) and incubate at Temperature37 °C DurationOvernight .
Note
C. rodentium has characteristic colony morphology of a red center and white rim (only on MacConkey, not on LB: the overnights are grown on LB, colonies are 'normal' buff color).

Incubation
Overnight
On day 2, pick one colony and inoculate Amount3 mL of LB (no antibiotic, regardless of strain) in a labelled culture tube and shake at Shaker220 rpm, 37°C, 17:30:00 .

Incubation
Mix
On day 3, mix Amount250 µL of 50% glycerol with Amount250 µL of overnight culture (final of 25% glycerol) in a labelled cryotube, vortex and freeze at Temperature-80 °C .
Note
Ensure to clearly label vials and box with pathogen, strain, antibiotic resistance, and date.


Mix
Temperature
Prepare Citrobacter Inoculum and In Vivo Infection
Prepare Citrobacter Inoculum and In Vivo Infection
On day 1, streak Citrobacter rodentium wild-type or chloramphenicol-resistant DBS100 strains from Amount1 mL frozen stocks on LB agar plates (with or without chloramphenicol) and incubate at Temperature37 °C DurationOvernight .
Incubation
Overnight
On day 2, pick one colony and inoculate Amount10 mL of LB (no antibiotic, regardless of strain) in a labelled glass culture tube (prepare in duplicate) and shake at Shaker220 rpm, 37°C, 17:30:00 .

  • The day of the infection, spin down the inoculum at Centrifigation4000 rpm .
  • Resuspend in Amount1 mL of plain LB media.
  • Pool total Amount6 mL into twist cap 15ml tube.
  • Place the tube of dose in a sealed bag for transport to the BCL-2 and pathogen transport container Saf-T-Pak.
Centrifigation
Pipetting
The day of the infection, gavage mice with Amount100 µL of overnight culture per mouse using a 22G gavage needle.
Note
See Substance administration SOP. The average dose is 2-3x109 Citrobacter rodentium per Amount100 µL .


Perform 1 in 10 serial dilutions of dose injected into mice, and plate 10-6 and 10-7 dilutions on LB plates. Take Amount100 µL bacteria (vortex very well) in Amount900 µL saline. Plate Amount100 µL per plate.

The next day, record CFU counts, and calculate actual dose infected in mice.
Monitor Citrobacter-Infected Mice and Time Point Studies
Monitor Citrobacter-Infected Mice and Time Point Studies
1h 11m
1h 11m
Record mouse weight daily. Monitor for clinical signs and symptoms up to 30 days post-infection. Mice must be humanely euthanized when they reach clinical endpoints:

  • Loss of >20% bodyweight
  • Body score condition <2
  • Loose stools, bleeding from anus, ruffled fur, lessened activity/lethargy, dehydration, piloerection, and sunken eyes and abdomen.
At specific time points post infection (day 3, 6 and 9), collect feces and/or euthanize mice.
Note
See Euthanasia and mouse necropsy SOP.

To collect feces:

  • Place individual mice in an empty cage (no bedding) for on average Duration00:10:00 (can take up to 2 hours) and collect 4 fecal pellets per mouse.
  • Weigh a 2ml tube with beads before and after adding fecal pellets and record weight of feces.
  • For highly susceptible animals (ex. C3H mice have no feces on day 9 post-infection).

10m
To prepare 2ml V-bottom cryotubes with beads:

  • add beads to individual cryotubes, tighten lid, place tubes with beads in a glass beaker, cover with aluminum foil and autoclave.
Add Amount1 mL PBS to each tube and incubate for Duration01:00:00 at Temperature4 °C to soften stool.

1h
Incubation
Pipetting
Homogenize fecal pellets using MagnaLyser at speed Centrifigation6000 rpm, 00:01:00 .

1m
Centrifigation
Plate undiluted to 10-6 dilutions (variable) on MacConkey agar supplemented with chloramphenicol to determine bacterial load.
Keep samples TemperatureOn ice in the cold room, you may need to replate some the next day.

The next day, count and record CFU per gram feces.