Lb lennox medium

Swabs from cloacal and fecal samples were incubated overnight in LB medium (Lennox) (Carl Roth, Karlsruhe, Germany) containing 2 µg/mL cefotaxime (VWR International GmbH, Darmstadt, Germany) at 37 °C while shaking at 200 rpm and subsequently cultivated on CHROMagar Orientation plates (MAST Diagnostica, Reinfeld, Germany) supplemented with 2 µg/mL cefotaxime at 37 °C. Putative cefotaxime-resistant E. coli colonies were identified based on colony morphology (pinkish colonies) and subjected to further cultivation until pure cultures were obtained. Pure cultures were used for further verification and characterization.

Based on a previous high‐throughput screening on 146 soil isolates (Tyc et al., 2014), a Gram‐negative strain, Burkholderia sp. AD24 (Beta‐proteobacteria) and a Gram‐positive strain, Paenibacillus sp. AD87 (Firmicutes) were selected for this study. The bacterial isolates were pre‐cultured from −80°C glycerol stocks on 1/10th TSBA (Garbeva and de Boer, 2009) for three days at 24°C. Two indicator bacteria, Escherichia coli WA321 and Staphylococcus aureus 533R4 were used as target bacteria to detect the production of antimicrobial compounds (Meyer and Schleifer, 1978; Tyc et al., 2014). The indicator bacteria were pre‐cultured from −80°C glycerol stocks on LB‐A media (LB‐Medium Lennox, Carl Roth GmbH + Co. KG, 20 g l⁻¹ Merck Agar). The indicator bacteria were incubated overnight at 37°C prior application. All bacterial isolates are listed in Table S4.

For in vivo production of dsRNAs, 275 mL of lysogeny broth (LB-Lennox) medium (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) supplemented with 100 µg/mL ampicillin (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and 12.5 µg/mL tetracycline (Fisher BioReagents, Geel, Belgium) were inoculated with 2.2 mL of an overnight culture of HT115 DE3 cells (F-, mcrA, mcrB, IN(rrnD-rrnE)1, rnc14::Tn10(DE3 lysogen: lavUV5 promoter -T7 polymerase, from the Caenorhabditis Genetics Center) transformed with L4440 plasmid encoding either a dsRNA against eGFP (480 bp) or Aae beta-tubulin (AAEL002851) (800 bp). Cells were grown at 37 °C and 180 rpm until the optical density measured at 600 nm (OD₆₀₀) reached 0.4 or 0.8. dsRNA production was then induced via addition of isopropyl-β-D-thiogalactopyranoside (IPTG) (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) to a final concentration of 0.4 mM and cells grown for another 4 h. Cells were harvested at 4 °C for 15 min at 3214 rcf, resuspended in clean LB-Lennox medium, aliquoted in batches of 10 OD or 2 OD in 2 mL-tubes, collected via centrifugation and stored at −80 °C until further use.