109(a) Suspend the CP in 0.9% saline to 10^8 CFU (colony form-
ing units)/mL using a colorimeter (see Note 10).
(b) Administer the suspension to the mice as 0.25 mL through
a stainless steel orogastric feeding tube.
(c) Leave the mice subsequently for 3 h, before the first dose
of antibiotic is administered subcutaneously as a maximum
of 0.25 mL as according to good practice [ 31 , 40 ] (see
Notes 11 and 12 ). To effectively mimic the serum antibi-
otic concentrations achieved in humans (on standard
doses), all mouse doses for antibiotic administration are
calculated based on human dose (in mg/kg of body
weight) from previously published mouse-studies or
PK-studies performed at Statens Serum Institut [ 22 , 29 ,
31 , 41 – 45 ] (see Note 13).
(d) Mice droppings are collected into individual 15 mL
Nunc™ tubes for each cage (feces belongs to two mice)
using disposable and sterile tweezers (see Note 14). Collect
a total of 0.5 g of feces from each cage.- On day 2 and 4
(a) The mice inhabiting the cage are moved to a clean cage
preceding sampling of feces.
(b) Individual mice droppings are collected into a 15 mL
Nunc™ tube as described on day 1.
(c) The antibiotics are administered subcutaneously. - On day 8
(a) The mice are sacrificed.
(b) Individual mice droppings are collected into a 15 mL
Nunc™ tube as described on day 1. - Three different plates are used for identification of ESBL-
producing E. coli, Gram-positive bacteria and Bacteroides,
respectively (see Notes 1– 3 ). - On the day of collection (preferentially done within 1 h of
sampling), dissolve the 0.5 g of individual feces samples in
5 mL of saline, vortex and leave for 1 h. - Dilute the samples tenfold in saline by serial passage of 100 μL
sample to 900 μL of saline a total of six times (dilutions 10−^1 to
10 −^6 ). 20 μL of each solution is spotted on duplicate plates of
each of the different selective agars (six plates in total)—
creating a circle of spots with the undiluted solution placed in
the middle (see Note 15). The spots cannot touch each other.
Leave the plates to dry on the table until the spot is completely
dry before moving to culturing. Culture the plates under
appropriate conditions for 18–20 h (37 °C) (see Notes 1– 3 ).
3.2 Determining
Colony Forming Units
In Vivo Antibiotic Selection