Luria broth

The antimicrobial activity of flours from oilseed cakes was determined for the following microorganism strains: (1) Gram-positive bacteria (G+) Staphylococcus aureus (ATCC 9538), Bacillus subtilis (ATCC 6633), Enterococcus faecalis (ATCC 29212) and Enterococcus hirae (ATCC 10542); (2) Gram-negative bacteria (G−) Escherichia coli (ATCC 10536), Vibrio harveyi (ATCC 12126) and Pseudomonas aeruginosa (ATCC 15442); and (3) the yeast Candida albicans (ATCC 10231). All microorganisms were obtained from the culture collection of the Department of Biotechnology and Food Microbiology (WUELS, Wrocław). Bacteria were grown in Luria broth (Sigma, Germany; containing 10 g/L tryptone, 5 g/L yeast extract and 5 g/L NaCl) at 37 °C; yeast was grown in YPD broth (Sigma, Germany; containing 20 g/L bacteriological peptone, 10 g/L yeast extract and 20 g/L glucose) at 30 °C. Agar was added to the medium at a concentration of 2% when necessary. To prepare an inoculation culture for the agar diffusion method, the cultures were grown in a 0.1 L flask containing 10 mL of proper medium, on a rotary shaker at 37 or 30 °C at 180 rpm for 48 h. Cells were washed in saline solution and adjusted to OD600 = 1.

The bacterium E. coli O86:B7 (ATCC 12701) expresses high levels of α-Gal on its surface [7 (link),8 (link)], which is not the case for E. coli BL21 (DE3, Invitrogen, Carlsbad, CA, USA) [7 (link)]. The E. coli strains were grown on 50 mL of Luria Broth (Sigma-Aldrich, St. Louis, MO, USA), incubated at 37 °C with vigorous shaking overnight, washed twice with PBS, centrifuged at 4000× g for 5 min at 4 °C and re-suspended at a concentration of ~1 × 10¹⁰ colony-forming units (CFU)/mL of PBS. For oral administration of bacteria, 7-day-old turkeys (n = 20) received E. coli strain O86:B7 (n = 10) or E. coli strain BL21 (n = 10) (~1 × 10⁹ CFU in 100 µL of PBS) via oral gavage at days 0, 1, 3, 7, 8, 9, 14, 15 and 16. All reagents used for bacterial preparation were apyrogenic.

E. coli and Klebsiella spp. displaying an ESBL or AmpC phenotype were screened for ESBL-and pAmpC encoding genes by multiplex PCR. The primers used for PCR are listed in Additional file 1: Table S1. PCR was carried out as previously described [26 (link)]. PCR products were separated and visualised using the Bioanalyzer DNA 1000 system (Agilent Technologies, Santa Clara, US) according to the manufacturer’s protocol.
Isolates with positive PCR results were selected for whole-genome sequencing (WGS) to further characterise the mechanism of resistance. Cells were grown over night in Luria Broth (Sigma Aldrich, St. Louis, US) with shaking at 35 °C, and DNA was extracted from 1 ml culture using the Wizard Genomic DNA Kit (Promega, Madison, US) according to the manufacturer's instructions. Quantification of total DNA was performed using a Qubit fluorometer (Life Technologies, Waltham, US), with broad range or high specificity reagents as appropriate. The sequencing libraries were prepared with the Nextera XT DNA Sample Prep Kit (Illumina, Eindhoven, the Netherlands) and sequencing was performed using a MiSeq sequencer (Illumina) in a 2 × 150-bp paired-end run. AMR genes were identified from WGS data using ResFinder [27 (link)] and Arg-annot [28 (link)].

An Escherichia coli (Ec 039) strain and a Legionella pneumophila serogroup 1 (Lp1 008-GFP) GFP-modified strain were used [41 (link),42 (link)]. They were stored at -80°C in Cryobank tubes (Mast Diagnostic, France). After thawing, Lp1 008-GFP and Ec 039 were plated, respectively, onto BCYE agar (Buffered Charcoal Yeast Extract, SR0110 C, Oxoid, France) and Luria Broth (LB) agar supplemented with chloramphenicol (Sigma Aldrich, France) at 8 mg/mL (for GFP plasmid selection) for 24 h (Ec 039) or 72 h (Lp1 008-GFP) at 37°C. They were then re-plated onto the same medium and incubated at 37°C for another 24 h (Ec 039) or for 3 days (Lp1 008-GFP). These cultures were then used to achieve a 10-mL calibrated suspension (CS) in sterile normal saline (0.9% NaCl) water. The final concentrations tested were 2x10⁶ and 2x10⁵ CFU/mL for Legionella and 2x10⁶ CFU/mL for E. coli.

Biotin N-hydroxysuccinimide ester and streptavidin were from Sigma-Aldrich. Goat anti-mouse IgG and IgM polyclonal antibodies were from Jackson ImmunoResearch. Rat anti-mouse IgG1, IgG2a and IgG2b antibodies were from AbD Serotec. Goat anti-mouse IgA antibodies were from CliniSciences. Sandwich ELISAs were performed with MaxiSorp 96-well microtiter plates (Nunc, Thermoscientific), and all reagents were diluted in Enzyme ImmunoAssay (EIA) buffer (0.1 M phosphate buffer [pH 7.4] containing 0.15 M NaCl, 0.1% bovine serum albumin [BSA], and 0.01% sodium azide). AEBSF (serine protease inhibitor) was from Interchim. Dialysis membranes were from Spectra/Por. Cholera Toxin and Luria Broth were from Sigma. PBS was from Gibco by Life Technologies.