Cefotaxime

Nutrient media were prepared on the basis of commercial Murashige and Skoog Basal Salt Mixture (MS) (MS5524, Sigma–Aldrich, Saint Louis, MO, USA). Media for co-cultivation, regeneration, shoot elongation, and rooting were prepared by supplementing the base MS5524 medium with the components listed in Table 1.
The freshly prepared medium was autoclaved at 121 °C and 220 kPa for 15 min and dispensed in 50 mL portions into sterile polycarbonate GA-7-3 culture vessels (Sigma-Aldrich, Saint Louis, MO, USA) and in 10 mL portions into 150 × 25 mm glass tissue-culture tubes (Z681784, Sigma-Aldrich, Saint Louis, MO, USA) with plastic vented closures. Kanamycin sulfate (A1493, AppliChem, Barcelona, Spain) and cefotaxime (Lekko, Volginskiy setl., Russia) were added to the air-cooled autoclaved medium (55 °C) to prevent thermal degradation of the antibiotic.

Before starting a trial, the complete experimental setup was tested for the absence of ESBL-/AmpC- producing bacteria after the introduction of the litter, feed, and water into the experimental room. Walls/doors, floor, table, heating lamps, feeding-, and drinking troughs were sampled with sterile and moistened gauze swabs. For moistening, 5 ml PBS were used. In addition, 5 g of litter, and feed were collected. Following the sampling, the gauze swabs, litter, and feed were transferred into 50 ml LB broth in each case and incubated for 24 h at 37°C before 10 μl were streaked out on chromogenic agar containing 2 μg/ml cefotaxime (AppliChem, Darmstadt, Germany) and incubated for 24 h at 37°C.
Directly after hatching, the absence of ESBL and AmpC- producing bacteria in the egg shells was confirmed. Five egg shells were crushed, transferred into 50 ml LB broth and incubated for 24 h at 37°C before 10 μl were streaked out on chromogenic agar containing 2 μg/ml cefotaxime and incubated for 24 h at 37°C.
The absence of ESBL and AmpC in the 1 day-old broiler chickens was confirmed by cloacal swabs of all individually tagged animals. Swabs were transferred into reaction tubes containing 500 μl PBS and were thoroughly vortexed. Fifty microliter were streaked out onto chromogenic agar containing 2 μg/ml cefotaxime and incubated for 24 h at 37°C.

The sample processing was identical to the one which was described in our seeder-bird colonization model [12 (link)]. Chromogenic orientation agar (CHROMagar Orientation, Mast Diagnostica, Reinfeld, Germany) was used for a reliable identification of the E. coli colonies. To confirm the ESBL-/ pAmpC- absence in the experimental room and the newly hatched broiler chickens, the agar was supplemented with two μg/ml cefotaxime (AppliChem, Darmstadt, Germany). In order to process all other samples, a set of four chromogenic agar plates which has proven suitable for our study was used. The total count of E. coli colonies was determined using an agar plate without selective media (positive control). For the detection of the ESBL- E. coli 10716, one plate was supplemented with two μg/ml cefotaxime and four μg/ml enrofloxacin (Sigma- Aldrich, Steinheim, Germany). For the detection of the pAmpC- E. coli 10717, one agar plate was supplemented with two μg/ml cefotaxime and seven μg/ml colistin (Carl Roth, Karlsruhe, Germany). The fourth agar plate contained all three antibiotics in the given concentrations (negative control). All samples were incubated for 24 h at 37°C. Every untypical E. coli colony morphology was further analyzed using MALDI- TOF (MALDI Microflex LT^® and Biotyper database^®; Bruker Daltonics, Bremen, Germany).