Nutrient medium

All samples collected during the experiments were tested for the presence of applied microorganisms, and analyses were carried out under a laminar flow hood using standard microbiological methods. To evaluate the presence and viability of the different microorganisms in the samples (viable and culturable), 100 µL of suspension from each sample was inoculated onto selective media for each microorganism: XLT-4 Agar (Xylose Lactose Tergitol 4, Scharlau, Barcelona, Spain) for S. Typhimurium detection; Baird Parker Agar (Scharlau, Barcelona, Spain) for S. aureus detection; Cetrimide Agar (Scharlau, Barcelona, Spain) for E. aerogenes detection; and Sabouraud Chloramphenicol Agar (Scharlau, Barcelona, Spain) for A. brasiliensis detection. Culture plates were incubated for 24–48 hours at 37 °C. After incubation, suspected bacteria colonies were streaked into a nutrient medium (Scharlab, S.L., Barcelona, Spain) and incubated at 37 °C for 24 hours. Then, API-test (Biomerieux, S.L., Barcelona, Spain) was performed to confirm the bacteria obtained. Filamentous fungi were identified on the basis of macroscopic and microscopic morphologic features (Figure 2).

First, cecal content was removed and homogenized. Then, pools of six animals from the same experimental group were prepared: 5 pools from day-old-chicks (30 samples), 10 pools from animals in CFC at mid-period (60 samples), 10 from animals in IFC at mid-period (60 samples), 10 pools from animals in CFC at the end of the growing period (60 samples) and 10 pools from animals in IFC at the end of the growing period (60 samples). The pools’ content was cultured directly onto a Coliform Chromogenic agar (Scharlab, S.L., Barcelona, Spain) in duplicate, and agar plates were incubated at 37 ± 1 °C for 24 h. After incubation, suspected colonies were streaked onto a nutrient medium (Scharlab, S.L., Barcelona, Spain) and incubated at 37 ± 1 °C for 24 h. Then, API-20E test (Biomerieux, S.L., Barcelona, Spain) was performed to confirm E. coli.