Macconkey agar

Feces were sampled from the donor sows 2 months before the experiment, at Day 14 and at Day 27. Feces from the lactating sows and from their piglets were sampled at Day 14 and Day 27 (weaning). Feces (5 g) from each animal were blended, homogenized in 45 mL of peptone water including 30% glycerol, and stored at −80 °C until use. Ten-fold serial dilutions of the homogenate were prepared, and 100-µl samples of the dilutions were spread on MacConkey agar (Sigma, M8302) in order to obtain ten isolates of Enterobacteriacae. The antibiotic susceptibility of Enterobacteriacae isolates was tested by plating a 24 h pure culture onto Mueller–Hilton agar alone or supplemented with 16 µg/mL tetracycline or 128 µg/mL sulfamethoxazole, according to the E. coli epidemiological cut-off values defined by Eucast [26 ]. Every test included a resistant and sensitive control strain to certify that the antibiotic was active. The proportion of sulfonamide- or tetracycline-resistant Enterobacteriacae was calculated for each animal.

In vitro conjugation experiments were conducted in liquid medium using the plasmid-free rifampicin-resistant E. coli recipient strain CV601 GFP at a donor:recipient ratio of 1:1 as previously described (Blau et al., 2018 (link)). Transconjugants were selected after incubation at 37°C for 24–48 h under selective conditions on lysogeny broth (LB) agar (Sigma-Aldrich, St. Louis, MO, United States) containing colistin sulfate (1 μg/ml) and rifampicin (200 μg/ml) (w/v). Isolates that did not yield transconjugants were further subjected to filter mating assays with the rifampicin-resistant, lactose-negative E. coli recipient strain W3110 at a donor:recipient ratio of 10:1 (Kieffer et al., 2017 (link)). The selection of transconjugants was done on MacConkey agar (Sigma-Aldrich, St. Louis, MO, United States) containing colistin sulfate (1 μg/ml) and rifampicin (200 μg/ml) after incubation at 37°C for 24–48 h under selective conditions. Potential transconjugants were subjected to PCR to confirm the presence of the mcr genes. Those transconjugants obtained with E. coli CV601 as recipient were additionally examined for GFP fluorescence using fluorescence microscope Axio Scope.A1 (Carl Zeiss Microscopy GmbH, Jena, Germany).

For P. aeruginosa isolation, 50 μL of the sputum samples was inoculated onto MacConkey Agar or Cetrimide agar (Sigma-Aldrich, St. Louis, MO, USA) incubated at 37°C for up to 4 days. Lactose-negative colonies on MacConkey Agar were selected and then subcultured onto a 5% sheep blood agar (Fisher Scientific, Dickinson).
To identify the BCC, we used Burkholderia cepacia-selective agar and cultured according to laboratory recommendations at 37°C for 24 to 72 h. Bacterial isolates were identified by using Vitek-2 (bioMérieux, France) according to the manufacturer’s instruction with a Gram-negative identification card.
P. aeruginosa bacterial density was assessed by serial 1:10 dilutions in PBS (pH 7.0) and was performed by plating 100 μL of sputum mix onto MacConkey Agar plates (Oxoid). These plates were incubated at 37°C aerobically for 48 h to isolate P. aeruginosa. Bacterial density was reported as log₁₀ CFU per milliliter of sputum.

Rectal swabs were placed in 5 ml of tryptic soy broth (HiMedia, Mumbai, India) supplemented with a 10 μg meropenem disc (Oxoid, Cambridge, UK) for overnight selective enrichment at 37°C (CDC, 2009 ). A loopful from the enrichment broth was streaked onto MacConkey agar supplemented with meropenem (1 mg/L) (Sigma-Aldrich, Seelze, Germany) and then incubated at 37°C for 24 h. Subsequently, the agar plates were examined for presumptive carbapenemase-producing Enterobacterales (CPE), which were identified based on their growth as either lactose-fermenting or lactose-nonfermenting colonies (Marques et al., 2019 (link)).

E. coli J62 lac pro trp his pFlac::Tn3 (Amp^R) and Salmonella Enteritidis P125109^{51 (link)} from which the virulence plasmid had been eliminated^{52 (link)} and the pFlac::Tn3 introduced (Amp^R) were used as host for phage MS2. A rifampicin resistant mutant of the Salmonella strain (Amp^R, Rif ^R) was used for the in vivo experiments. A collection of plasmid-containing wild-type AMR E. coli strains isolated from poultry^{37 (link)} were selected for host range, phage MS2 sensitivity and plasmid loss experiments (Table S1). E. coli J62 Rif ^R was used as recipient strain for plasmid transfer rate assessment.
Luria Bertani (LB) broth and agar (1%, Sigma) was used for routine culture of the strains. Viable counts of E. coli J62 pFlac::Tn3 (Amp^R) and AMR strains were determined by plating dilutions on MacConkey agar (Sigma) with or without antibiotics. The same was done for S. Enteritidis P125109 pFlac::Tn3 (Amp^R, Rif ^R) counts, using Brilliant green agar (Sigma) supplemented with the appropriate antibiotic. For dilution purposes Phosphate-Buffered saline (PBS) buffer was used.