After reviewing the results of the previous mouse competition experiment, where the ancestral non-bioluminescent ICC169 strain was competed with the adapted bioluminescent strains W1 (adapted to mice hosts receiving untreated food/water) and N4 (adapted to mice hosts receiving low-dose nalidixic acid treatment), a further mouse competition experiment was designed and performed. Three more adapted isolates from mice receiving nalidixic acid (N1, N2, and N3) were chosen, and all competition mouse groups received low-dose nalidixic acid. As before, the EP20 of each adapted isolate was chosen (ie the effective passage number 20).
Three groups of 6 mice each were used:
1) C57BL/6 mice, receiving autoclaved food/water, with 10 ug/mL nalidixic acid in the drinking water, infected with N1 and ICC169.
2) C57BL/6 mice, receiving autoclaved food/water, with 10 ug/mL nalidixic acid in the drinking water, infected with N2 and ICC169.
3) C57BL/6 mice, receiving autoclaved food/water, with 10 ug/mL nalidixic acid in the drinking water, infected with N3 and ICC169.
ICC169, W1, and N4 cultures were grown overnight from frozen stocks in LB with no selection. Optical density at 600 nm was determined and cultures were matched in order to ensure as similar doses of each strain as possible. The cells were then pelleted and resuspended in sterile PBS, resulting in a 10x concentrated mixture. The cultures were then mixed in the 1:1 ratios (ICC169 with W1 for group 1, and ICC169 with N4 for groups 2 and 3) and used to artificially infect mice.
Mice were infected via oral gavage at the following doses (ratios determined via plating onto 50 ug/mL nalidixic acid and 50 ug/mL kanamycin plates):
169 (to group 1): 1.06e9 CFU
N1 (to group 1): 4.70e8 CFU
169:N1 ratio: 69.3:30.7
169 (to group 2): 7.53e8 CFU
N2 (to group 2): 3.73e8 CFU
169:N2 ratio: 66.9:33.1
169 (to group 3): 8.15e8 CFU
N3 (to group 3): 1.87e7 CFU
169:N3 ratio: 97.8:2.2
For an undetermined reason (possibly pipetting error), the ratio of wildtype to N3 in group 3 was dramatically in favour of the wildtype, with only ~2% of bacteria infecting the mice belonging to the adapted population.
As before, the mice were monitored for wellbeing and weightloss using standardised animal monitoring sheets, with stool samples taken to determine the ratio of strains shed in faeces. On day 7, a further 6x mice for each treatment group were housed in ‘dirty’ 1-day old cages from the infected mice. These were followed in a similar manner as before in order to determine whether the adapted strains are more readily transmitted from contaminated stools.
Raw data: Bacterial counts
Group 2 (infected with 169/N2) failed to transmit either the wildtype or the adapted strain to the ‘transmission’ (uninfected) mice. Due to the incorrect dosing of group 3, no adapted isolates could be detected in the transmission mice, and the Citrobacter rodentium populations isolated from the gavaged mice quickly favoured the wildtype due to its overwhelming initial number advantage.