Tag Archives: Evolution in Action

HANNAH 12/11/2014

From 3/07/2014 to 7/11/2014 we sought to determine the mutation rate of the mouse-adapted strains compared to their ancestors (ICC169 and ICC180) using spontaneous resistance to two antibiotics which require a single point mutation for resistance to occur as a proxy.

The strains were tested for sensitivity to rifampicin and streptomycin before continuing.

Optomisation of the assay was performed until the final protocol was designed:

Bacteria were grown from freezer stocks in 1.5 mL of rich media (LB) in 5mL yellow capped tubes overnight (with shaking). 15 uL of the overnights were then inoculated into fresh 15mL of LB in 50mL falcon tubes and incubated overnight again. 400 uL of the overnight was then plated onto LB plates containing 80 ug/mL rifampicin, and 20 uL of a 1/10 dilution of the overnight was plated onto LB plates containing 20 ug/mL streptomycin. Samples were also plated onto plain LB agar to enumerate. Rates of resistance was determined as a percentage of resistant colonies to the total number of cells.

Raw data: Spontaneous resistant colonies to rifampicin and streptomycin

 

Hannah 21/03/2014

From 11/03/14 through to 14/03/14, a caterpillar assay was performed. Four different doses of the wildtype strains (ICC169 and ICC180) and of four mouse-adapted isolates (W1, W3, W5, and N4) were used to infect caterpillars in groups of 10, which were then housed at 37 degrees Celsius and monitored twice daily using the Caterpillar Health Index scoring system.

As with previous experiments, the EP20 (effective passage number 20) of all strains was used. Kanamycin-selected isolates were used (ie not from mixed culture frozen stools).

Raw data: CHI scores and doses

Hannah 25/02/2014

From 20/01/2014 to 5/02/2014, a mouse competition assay was performed as described previously (see here and here).

Briefly, overnight cultures of the wildtype non-bioluminescent strain ICC169, and the adapted isolates of interest (N2, N3, and N5) were grown in rich LB broth without antibiotic selection. The cultures were spun down and resuspended in sterile PBS to result in a 10x concentrated solution. ICC169 was mixed in a 1:1 ratio with each of the adapted isolates (169/N2, 169/N3, and 169/N5) and the mixed cultures were used to infect 3 groups of 6 mice. The mice had received 10 ug/mL nalidixic acid in their drinking water for 1 day prior, and remained on antibiotic treated water for the duration of the experiment. Stool samples were taken daily from each individual animal and plated onto selective agar to determine proportion of C. rodentium strains shed in faeces. Animals were imaged twice per week. At the peak of infection, the groups of mice were put into clean cages for 24-hours, then moved into new cages. The 1-day old cages were then filled with fresh groups of uninfected mice, in order to investigate disease transmission dynamics.

Bacterial doses to groups of 6 mice are as follows
Group 1 (169/N2)
169: 1.80e9
N2: 2.90e9

Group 2 (169/N3)
169: 1.27e9
N3: 3.17e9

Group 3 (169/N5)
169: 1.60e9
N5: 1.53e9

Raw data: Bacterial counts

Hannah 25/02/2014

From 20/01/2014 to 24/01/2014, a further caterpillar experiment was designed and performed. Caterpillar Health Index scores were taken of caterpillars at each time point. The N isolates (adapted isolates taken from mice receiving 10 ug/mL nalidixic acid treatment) and W isolates (adapted isolates taken from mice receiving plain autoclaved food/water) were run through the caterpillar assay in single infections, with the aim of investigating time to death and disease symptoms over time.

As with the complementary mouse experiments, the EP20 (effective passage number 20) of all strains was used. Kanamycin-selected isolates were used (ie not from mixed culture frozen stools).

The strains used and their dose per caterpillar is as follows:

ICC169: 7.08e7
ICC180: 8.21e7
W1: 8.83e7
W2: 7.50e7
W3: 1.05e8
W4: 8.21e7
W5: 8.67e7
N1: 8.46e7
N2: 9.00e7
N3: 8.38e7
N4: 9.38e7
N5: 5.08e7

Raw data: CHI scores

Hannah 17/01/2014

Repeat of in vitro competition assay comparing ancestral Citrobacter rodentium to mouse-adapted isolates (pilot experiment here). Performed as described previously, with the following adjustments – all mouse-adapted strains were used (N1-5 and W1-5), and two independent cultures were set of each strain (ie 2 biological replicates each time). The experiment was performed on two separate days (4x biological replicates total).

Raw data: Bacterial counts and competition indices for replicates 1 and 2

Raw data: Bacterial counts and competition indices for replicates 3 and 4

Hannah 10/01/2014

To investigate in vitro competitive fitness of mouse adapted Citrobacter rodentium compared with the wildtype strain, the Nx EP20 strains (mouse adapted C. rodentium isolates from mice receiving nalidixic acid, effective passage number 20) were competed with the ancestral non-bioluminescent ICC169 strain in defined minimal media. ICC180 (the bioluminescent ancestor of the Nx strains) was competed with ICC169 as a reference point.

 

Kanamycin-selected C. rodentium from mice receiving nalidixic acid (Nx strains) at effective passage number 20 were chosen for initial analysis. Km-selected Nx (1-5) EP20 and the ancestral ICC169 and ICC180 strains were grown overnight at 37 degrees Celsius in 1mL of LB. 20uL of ICC169 and 20uL of either ICC180, N1, N2, N3, N4, or N5 went into 1mL of defined minimal media and incubated for 24-hours. The starting ratio and end ratio after 24-hours of each strain was determined by plating onto nalidixic acid and kanamycin (both 50 microgram/mL) LB agar plates.

 

The data from the initial pilot experiment (only 1 tube each, no biological replicates) is shown below.
This will be repeated with 2x biological replicates of each tube, all 10 mouse adapted strains.
[EDIT 17/01/2014: Fixed calculation error and formatting.]

Hannah 16/12/2013

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.

Hannah 11/11/2013

In order to compare the adapted isolates in a manner best suited to their particular adaptations, a competition experiment in mice was performed. Three strains of Citrobacter rodentium were used in this experiment — ICC169, the nalidixic acid-resistant, non-bioluminescent ancestor of all adapted isolates; W1 [effective passage number 20/EP20], a nalidixic acid and kanamycin-resistant bioluminescent derivative of ICC169 which has ‘adapted’ to mice with normal commensal microbes; and N4 [EP20], a nalidixic acid and kanamycin-resistant bioluminescent derivative of ICC169 which has ‘adapted’ to mice with disrupted microbiota due to 10 ug/mL nalidixic acid treatment in their drinking water.

Three groups of 6 mice were used:
1) C57BL/6 mice, receiving autoclaved food/water, infected with a 1:1 ratio of ICC169:W1.
2) C57BL/6 mice, receiving autoclaved food/water, infected with a 1:1 ratio of ICC169:N4.
3) C57BL/6 mice, receiving autoclaved food/water, with 10 ug/mL nalidixic acid in the drinking water, infected with a 1:1 ratio of ICC169:N4.

 

Groups 1 and 2 would compare fitness of isolates ‘adapted’ to the two different conditions: ie, hosts with commensals (no antibiotics) and hosts with reduced commensal loads (receiving antibiotics).
Groups 2 and 3 would compare fitness of an isolate ‘adapted’ to a host with reduced commensals, in two different conditions (with and without antibiotics).

 

ICC169, W1, and N4 cultures were grown overnight from frozen stocks in LB with no selection. 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): 1.68e9 CFU
W1 (to group 1): 8.85e8 CFU
169 (to groups 2 and 3): 1.76e9 CFU
N4 (to groups 2 and 3): 1.04e9 CFU

 

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. Mice were also shaved and imaged twice weekly.

 

On day 8, 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.

 

At the end of the experiment (day 16 for the originally infected mice, and day 8 for the ‘transmission’ mice), serum and colon samples were taken and frozen for future analysis.

 

Hannah 18/10/2013

Growth curve of W1 and N4, with the ancestral strains ICC169 and ICC180. ICC169 is a spontaneous nalidixic acid resistant mutant of Citrobacter rodentium ICC168, while ICC180 is the bioluminescent derivative of ICC169 with kanamycin resistance. ICC180 is also the ancestor of all adapted strains. W1 and N4 are adapted isolates chosen for an initial pilot mouse experiment, scheduled to begin on the 21/10/2013. W1 and N4 have been chosen due to their improved growth in minimal media, and because neither strain ever failed to transmit between mice during the in vivo evolution experiment.

The strains were grown overnight in LB, washed once in PBS, and inoculated 1:100 in defined minimal media. Samples were taken from the cultures for enumeration and to measure bioluminescence.

Raw data: Bacterial counts and bioluminescence over time