Api 20e galleries

Isolation and identification of Salmonella spp. were performed according to ISO 6579 [18 ] using the media described in international standard protocols (Table 2).
Five suspected colonies from each Petri dish were streaked on Columbia blood agar (CBA) (Becton & Dickinson, Franklin Lakes, NJ, USA) and incubated at 35 °C for 24 ± 2 h. One isolated colony from each CBA plate was analyzed with a MALDI Biotyper^® Sirius System mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) using the extended direct transfer (eDT) procedure. One loop of biomass was transferred to a MBT96 polished steel BC target plate (Bruker Daltonik GmbH) spot. The air-dried sample spot was overlaid with 1 µL of a formic acid water solution (70% v/v) and, after air-drying, with 1 µL of matrix HCCA (α-cyano-4-hydroxycinnamic acid) solution (Bruker Daltonik GmbH). Target plates of the samples were analyzed, and spectra were acquired in positive ion mode in the m/z range 2000–20,000. As an alternative, one isolated colony from each CBA plate was streaked on triple sugar iron agar (TSI) (Biolife Italiana S.r.l., Milan, Italy). Positive TSI colonies were confirmed by biochemical analysis using API 20E galleries (bioMérieux, Marcy l’Étoile, France).

In the bacteriology laboratory of the Interdisciplinary Center for Medical Research of Franceville (CIRMF), the bacteria culture consisted of emulsifying some of the faecal specimens in sterile water and streaking a loopful of the resultant on MacConkey agar medium (MCA) (bioMérieux, France). After eighteen to twenty-four hours of incubation, several colonies (never more than two or three from one medium Petri dish), differentiated by structure and colour, were picked up and streaked in the same medium and incubated in the same conditions. A loop of liquid was removed from the cultures and streaked onto methylene eosin blue agar (EMB) plates (bioMérieux, France) for E. coli isolation. Plates were examined for the characteristic appearance of E. coli (metallic green sheen) and colonies with green metallic sheen were interpreted as E. coli isolates [117 (link),118 (link),119 (link)]. Purified green metallic colonies were subjected to biochemical identification using the Api 20 E galleries (bioMérieux, Marcy l’étoile, France) and the VITEK^® 2 Compact 15 system (bioMérieux, Marcy l’étoile, France). Isolates were confirmed by using phylogenetic group detection PCR as described by Clermont et al. [52 (link),53 (link)].

Wild birds (n = 200) were purchased alive on the Sousse market, Tunisia, between January and February 2022 during the legal hunting period. Birds were caught in Bizerte, Zaghouan, or Gabès (Fig. S1), and kept in aviaries for as short a time as possible, without being fed. The intestine of each bird was collected immediately after the death of the bird using a sterile scalpel and stored at −20°C. Intestinal content was emptied in 10 mL of Trypto-casein soy broth (Biokar), homogenized and incubated for 18–24 h at 37°C. Overnight cultures were inoculated on selective MacConkey agar plates supplemented with cefotaxime or imipenem (2 mg/L), for the detection of ESC- or CP-resistant Enterobacterales. One colony per morphology and per plate was picked up. Identification was performed using API20E galleries (bioMérieux).