Author Archives: Hannah Read

Hannah 2/07/2015

Performed 16/06/2015 – 30/06/2015. A mouse competition experiment competing the ancestral ICC169 strain with the ancestral ICC180, N3, and W5 strains.

As before: 15 mL overnight cultures of ICC169 (3x), ICC180, N3 and W5 were spun down and resuspended in 1.5 mL sterile PBS (resulting in 10x concentrated cultures), and cultures were mixed in a 1:1 ratio as follows: ICC169 with ICC180, ICC169 with N3, and ICC169 with W5. 200 uL of the mixtures were orally gavaged to groups of 6 C57BL/6 mice. For the ICC169/N3 group, the animals received 10 ug/mL nalidixic acid in their drinking water for 1-day prior.

Mice received fresh drugs on Mondays, Wednesdays, and Fridays, and were imaged using an IVIS Kinetic (Caliper LifeScience) machine on Mondays and Fridays. Stools were taken and plated daily to measure relative ratios of the strains shed. At day 6 post-infection, mice were housed in a fresh cage for 1-day. At day 7, mice were re-housed and the 1-day old ‘contaminated’ cages were used to house naive animals (groups of 6) in order to follow natural transmission of the strains. These animals similarly received antibiotic (in the case of the ICC169/N3 group) in the drinking water and were monitored the same as the original animals.

Raw data: Bacteria shed from mice

Hannah 2/07/2015

Performed 19/05/2015 – 3/06/2015: A mouse competition experiment competing the ancestral ICC169 strain with the ancestral ICC180, W4, and W5 strains.

As before: 15 mL overnight cultures of ICC169 (3x), ICC180, W4 and W5 were spun down and resuspended in 1.5 mL sterile PBS (resulting in 10x concentrated cultures), and cultures were mixed in a 1:1 ratio as follows: ICC169 with ICC180, ICC169 with W4, and ICC169 with W5. 200 uL of the mixtures were orally gavaged to groups of 6 C57BL/6 mice.

Mice were imaged using an IVIS Kinetic (Caliper LifeScience) machine on Mondays and Fridays. Stools were taken and plated daily to measure relative ratios of the strains shed. At day 6 post-infection, mice were housed in a fresh cage for 1-day. At day 7, mice were re-housed and the 1-day old ‘contaminated’ cages were used to house naive animals (groups of 6) in order to follow natural transmission of the strains. These animals were monitored the same as the original animals.

Raw data: Bacteria shed from mice

Hannah 2/07/2015

From the 20/04/2015 – 8/05/2015: In order to determine changes to the commensal bacteria during infection of C57bl/6 mice with the ancestral and ‘adapted’ Citrobacter rodentium, 6 groups of 6 mice (receiving nalidixic acid)were infected with each of the N (C. rodentium adapted to mice receiving low-dose nalidixic acid) strains or the ancestor ICC180. Stool samples were taken throughout the infection, ending at day 17 post-infection, and plated to monitoring bacterial shedding.

Stool samples were also taken at various time points (day 1, 3, 7, 10, 15, and 17 post infection) and snap frozen in a dry ice + ethanol bath for future 16s sequencing and RNA analysis.

Stool samples were also taken from the animals pre-infection both before receiving antibiotic and 24-hours after antibiotic to determine the effect of low-dose nalidixic acid on commensals.

Raw data: Bacteria shed from mice

Hannah 27/03/2015

Performed 9/03/2015 – 25/03/2015. A mouse competition experiment competing the ancestral ICC169 strain with the ancestral ICC180, W3, and W4 strains.

As before: 15 mL overnight cultures of ICC169 (3x), ICC180, W3 and W4 were spun down and resuspended in 1.5 mL sterile PBS (resulting in 10x concentrated cultures), and cultures were mixed in a 1:1 ratio as follows: ICC169 with ICC180, ICC169 with W3, and ICC169 with W4. 200 uL of the mixtures were orally gavaged to groups of 6 C57BL/6 mice. For the ICC169/ICC180 group, the animals received 10 ug/mL nalidixic acid in their drinking water for 1-day prior.

Mice received fresh drugs on Mondays, Wednesdays, and Fridays, and were imaged using an IVIS Kinetic (Caliper LifeScience) machine on Mondays and Fridays. Stools were taken and plated daily to measure relative ratios of the strains shed. At day 6 post-infection, mice were housed in a fresh cage for 1-day. At day 7, mice were re-housed and the 1-day old ‘contaminated’ cages were used to house naive animals (groups of 6) in order to follow natural transmission of the strains. These animals similarly received antibiotic (in the case of the ICC169/ICC180 group) in the drinking water and were monitored the same as the original animals.

Raw data: Bacteria shed from mice

Hannah 27/02/2015

Performed 2/02/2015 – 20/02/2015. A mouse competition experiment competing the ancestral ICC169 strain with the ancestral ICC180, W1, and W2 strains.

As before: 15 mL overnight cultures of ICC169 (3x), ICC180, W1 and W2 were spun down and resuspended in 1.5 mL sterile PBS (resulting in 10x concentrated cultures), and cultures were mixed in a 1:1 ratio as follows: ICC169 with ICC180, ICC169 with W1, and ICC169 with W2. 200 uL of the mixtures were orally gavaged to groups of 6 C57BL/6 mice. For the ICC169/ICC180 group, the animals received 10 ug/mL nalidixic acid in their drinking water for 1-day prior.

Mice received fresh drugs on Mondays, Wednesdays, and Fridays, and were imaged using an IVIS Kinetic (Caliper LifeScience) machine on Mondays and Fridays. Stools were taken and plated daily to measure relative ratios of the strains shed. At day 6 post-infection, mice were housed in a fresh cage for 1-day. At day 7, mice were re-housed and the 1-day old ‘contaminated’ cages were used to house naive animals (groups of 6) in order to follow natural transmission of the strains. These animals similarly received antibiotic (in the case of the ICC169/ICC180 group) in the drinking water and were monitored the same as the original animals.

Raw data: Bacteria shed from mice

Hannah 16/01/2015

A pilot growth curve of Citrobacter rodentium passaged through laboratory media (minimal A salts with 1% glucose) over the same number of days as the mouse-adapted strains passaged through animals (ie 140 days) was performed on the 15/01/2015.

Briefly, Citrobacter rodentium ICC180 strains taken at passage 140 (approximately 1,200 generations) and the ancestral ICC180 strain were revived from frozen stocks and grown overnight (2 biological replicates for each) in minimal A salts with glucose. 5uL of the overnight was added to 100uL of fresh media in 96-well plates in triplicate and incubated at 37 degrees Celsius, with OD600 and light levels measured hourly.

Only one line out of 6 independent lineages retained the ability to produce light (Tube 7), and so light levels could only be monitored from one of these samples.

Raw data: OD and RLU readings

Results show that C. rodentium strains adapted to minimal media conditions may have a slight growth advantage over their ancestor, particularly at the early growth phase. This is particularly apparent with the strain which retained light production (Tube 7).

Hannah 16/01/2015

Performed on from the 1/12/2014, a repeat of the infectious dose assay (first experiment here).

Treatment plan for the mouse groups as follows:
ICC180: 1e8, 1e6, and 1e5 (with nalidixic acid in drinking water)
ICC180: 1e8 and 1e6 (without antibiotic)

N4: 1e8, 1e6, and 1e5 (with nalidixic acid in water)
N4: 1e8 and 1e6 (no antibiotic)

N3: 1e8 and 1e6 (with nalidixic acid in water)

Raw data: Inoculation dose and bacterial shedding in stools

Results indicate that at the lowest dose on day 2 (~5 x 1e5 CFU, + nalidixic acid), the ancestral strain ICC180 is no longer infectious, while the N4 strain successfully infected all 4 animals, indicating a lowered infectious dose for this hypertransmissible mouse-adapted strain.

Hannah 12/11/2014

Performed from 30/10/2014 to 4/11/2014. Following the mouse transmission assays, which indicated that the N4 strain has an improved transmissibility phenotype (data here and here), we hypothesised that the mechanism of this may be shown as a reduced infectious dose compared to the wildtype and other mouse-adapted strains which did not develop the improved transmissibility phenotype.

Strains ICC180, N3, and N4 were chosen for comparative purposes; groups of 4 mice for each dose. 4 doses (10x concentration of an overnight, Neat of an overnight, 1/10 and 1/100 of an overnight culture).

Raw data: Infectious dose assay

Hannah 12/11/2014

From 17/09/2014 to 3/10/2014, a repeat of the mouse competition assays was performed to confirm the ‘hypertransmissibility’ phenotype of the N4 strain. Previously the N4 strain had undergone two concurrent mouse competition experiments (6x mice each, 1 group receiving 10 ug/mL nalidixic acid and 1 group without). This was repeated with 6x mice receiving antibiotic.

Similarly, the N3 strain (which appears to be ‘hypermutatable’) will be repeated in the mouse competition assay. Previously the N3 strain has shown no competitive advantage in the mouse competition model, and this is to be confirmed with a repeat experiment.

15 mL overnight cultures of ICC169 (2x), N3, and N4 were spun down and resuspended in 1.5 mL sterile PBS (resulting in 10x concentrated cultures), and cultures were mixed in a 1:1 ratio as follows: ICC169 with N3, and ICC169 with N4. 200 uL of the mixtures were orally gavaged to groups of 6 C57BL/6 mice which had received 10 ug/mL nalidixic acid in their drinking water for 1-day prior.

Mice received fresh drugs on Mondays, Wednesdays, and Fridays, and were imaged using an IVIS Kinetic (Caliper LifeScience) machine on Mondays and Fridays. Stools were taken and plated daily to measure relative ratios of the strains shed. At day 6 post-infection, mice were housed in a fresh cage for 1-day. At day 7, mice were re-housed and the 1-day old ‘contaminated’ cages were used to house naive animals (groups of 6) in order to follow natural transmission of the strains. These animals similarly received antibiotic in the drinking water and were monitored the same as the original animals.

Inoculation dose:
inoculationdose

Total dose of ICC169 to N3/ICC169: 3.73 e8 (45.5%)
Total dose of N3 to N3/ICC169: 4.47 e8 (54.5%)
Total dose of ICC!69 to N4/ICC169: 3.53 e8 (52.5%)
Total dose of N4 to N4/ICC169: 3.20 e8 (47.5%)

IE: approximately a 1:1 ratio of ancestor to evolved strain was achieved.

Raw data: Bacteria shed from mice

For transmission animals, when similar numbers were recovered on Nal and Km plates, Nal plates were put in the IVIS to check if any were not glowing (ie ICC169). If they were not, then numbers from nal and km plates were averaged and this used as number of ICC180 derivatives recovered.