Biotyper database

After selection of unsusceptible E. coli via agar dilution with cefotaxime containing MacConkey-agar (1 μg/mL), the species was confirmed using MALDI-TOF identification (MALDI Microflex ® LT and Biotyper ® database, Bruker Daltonics, Bremen, Germany). Identification of ESBL encoding genes of the CTX-M, TEM, SHV and CYM families was carried out by PCR according to the method published by Roschanski et al. [34 (link)].

Extended-spectrum β-lactamases/AmpC-producing E. coli strains derived from the strain collection of the Institute for Animal Hygiene and Environmental Health, Freie Universitaet Berlin. The isolates were obtained from 7 different pig farms (n = 104) and 22 turkey farms (n = 51) in Germany. Each isolate originated from an individual sample including feces, boot swabs, manure, air or dust. ESBL/AmpC E. coli were selected using MacConkey agar (Oxoid, CM 0115, Wesel, Germany) supplemented with 1 mg/L cefotaxime, followed by species confirmation using MALDI-TOF identification (MALDI Microflex LT and Biotyper database, Bruker Daltonics, Bremen, Germany). The presence of the β-lactamase genes bla_CTX, bla_TEM, bla_SHV and the CIT-type AmpCs (e.g., CMY-2) was confirmed by real-time PCR as described by Roschanski et al. (2014) (link).

Strains from the CSUR (Collection des souches de l’unité des Rickettsies, IHU Méditerranée infection, Marseille France) were used. Bacterial strains were identified by Maldi Toff mass spectrometry using the Bio Typer database (Bruker, Dresden, Germany). In a second time, they were cultured on 5% sheep blood-enriched Columbia agar (COS, BioMérieux, Marcy l’Etoile, France). After 18 h of incubation at 37°C, colonies were removed and suspended in NaCl at the required concentration. Three different strains of each species were used, thus Methicillin sensitive S. aureus (P6142, P2188 and P6141) and S. sanguinis (P8633, P760 and P2754). All strains were isolated from positive blood cultures.

The strains used in this study were selected from each group based on our previous results [9 (link)]. The two main selection criteria are the capacity of the strain to induce platelet activation and the profile toward the platelet inhibitory effect (Table 1).
The strains represent the following profiles: E. coli J53, platelet sensitive strain which induces platelet activation; E. coli K12, platelet resistant strain which induces platelet activation; and E. coli LH30, platelet resistant strain which does not induce platelet activation (Table 1).
Identification was confirmed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and the Biotyper database (Bruker, Dresden, Germany). Strains were grown at 37 °C in an overnight culture of Columbia agar +5% sheep blood (bioMérieux, Marcy l’Etoile, France). After 18 h of incubation at 37 °C, the colonies were removed and suspended in 0.9% NaCl medium to obtain the required concentrations: 1 × 10⁸ CFU (colony format units).

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).