Antibiotic Resistance Protocols (Methods in Molecular Biology)

107

The indigenous intestinal flora acts as a barrier against incom-

ing pathogens and overgrowth of opportunistic microorganisms

already present in the gut; known as colonization resistance.

However, alterations in the microbiota can allow for colonization,

with possible subsequent infection, and antibiotic treatment is

known to disturb the ecological balance of the indigenous micro-

flora [ 13 – 15 ].

Understanding how antibiotics select for an ESBL-producing

E. coli isolate is very important and here we present a mouse-

intestinal colonization model designed to explore this phenome-

non. The model can be used to study how antibiotics with varying

effect on the intestinal flora promote the establishment of the

multi-drug resistant E. coli—provided the antibiotic has no effect

on the administered bacterial strain harboring the relevant resis-

tance mechanism. Colonization is successfully investigated by sam-

pling and culturing stool during the days following administration

of antibiotics [ 29 ]. A precise identification of the bacterial strain

found in mice feces is of great importance to ensure that the isolate

found is in fact identical to the strain used for inoculation. For this

purpose random amplified of polymorphic DNA specifically devel-

oped for E. coli is applied. This method allows us to distinguish E.

coli with more than 99.95% genome similarity using a duplex PCR

method, based on short primers binding randomly to the DNA,

resulting in fingerprints of each strain on a gel [ 39 ].

2 Materials

1. Chromogenic agar plates: Discovery agar base (Oxoid) con-
   taining 32 μg/mL cefotaxime (Fresenius-Kabi) and 6 μg/mL
   vancomycin (Sigma-Aldrich) (see Note 1).


2. 5% Blood-agar plates containing 4 μg/mL gentamicin (SSI
   Diagnostica) (see Note 2).


3. Anaerobic agar plates containing 32 μg/mL gentamicin and
   16 μg/mL vancomycin (SSI Diagnostica) (see Note 3).


4. 0.9% saline: MilliQ water, 0.9% NaCl.


5. Freezing stock: Luria Broth (Sigma-Aldrich), 15% glycerol
   (Sigma-Aldrich).


6. Disposable needles (BD Medical).


7. Syringes (BD Medical).


8. Female albino outbred NMRI mice: 7–10 weeks old, weighing
   26–30 g (Harlan) (see Note 5).


9. Disposable, sterile tweezers (Unomedical).


10. Wide-bore 50–1000 μL pipette tips (Sartorius).


11. E-Gel® Electrophoresis System (Thermo Scientific).

In Vivo Antibiotic Selection