Antibiotic Resistance Protocols (Methods in Molecular Biology)

111

(e) Take a picture of the gel by your local UV gel electropho-

resis camera.

(f) The fingerprinting will differ in number of bands for dif-

ferent E. coli clones (less than 99.5% genome similarity)

(see Note 18).

4 Notes

1. The Chromogenic agar used can identify and quantify uro-
   pathogenic bacteria according to color (e.g., E. coli are red,
   Klebsiella spp. are blue, and Enterococcus spp. are green/blue).
   These agar plates are used to identify the colonizing pathogen
   and should therefore contain antibiotics with effect on the
   indigenous microbiota of the mouse, but without effect on the
   colonizing pathogen (the ESBL-producing E. coli).


2. Blood agar plates with gentamycin inhibit growth of any sus-
   ceptible E. coli and are therefore used to evaluate antibiotic
   impact on the Gram-positive flora of the mice gut. Gentamicin
   is used since the colonizing ESBL-producing E. coli is suscep-
   tible to gentamicin.


3. The anaerobic agar plates are used to evaluate the impact of
   antibiotics on the population of Bacteroides. These plates must
   be cultured under anaerobic conditions. If no anaerobic cham-
   ber is available, culturing of anaerobic spp. can be performed
   in sealed jars with anaerobic atmosphere, as created in GASPAK
   EZ containers by AnaeroGen® (Oxoid) [ 46 ].


4. We performed all experiment at Statens Serum Institut (SSI) in
   Copenhagen, Denmark. All animal experiments must be
   approved by the local Centre for Animal Welfare and carried
   out at approved facilities by trained personal with required
   certificates.


5. All mice should be from the same litter.


6. No alterations of the intestinal flora are induced prior to the
   study, i.e., no antimicrobial treatment is administered.


7. It is advised to perform experimental studies to ensure that the
   specific mouse-intestinal colonization model produces reliable
   and reproducible results. For our experimental studies, we
   used nine groups, with each group receiving one antibiotic
   only. Each group consisted of two cages with two mice each.
   For all studies, we included a control group.


8. The mouse intestinal colonization can be applied on isolates
   with other resistance mechanisms than ESBL. This requires
   that the applied colonizing isolate is resistant to the drugs
   administered and that the isolate chosen as colonizer, must be

In Vivo Antibiotic Selection