Amoxicillin

A total of 240 1-day-old chicks were randomly divided into 24 groups of 10 animals. Following the instructions of the manufacturer, different groups were treated with different antibiotics, which included ceftiofur (the dose was 0.1 mg per chicken by intramuscular injection at 1 day old; the withdrawal time is 3 days; Zoetis, New Jersey, USA), amoxicillin (amoxicillin was administered by drinking water for 5 days; the dose was 10 g/L water, and the withdrawal time is 7 days; Merck, New Jersey, USA), enrofloxacin (enrofloxacin was administered by drinking water for 5 days; the dose was 0.75 ml/L water, and the withdrawal time is 8 days; Bayer, Leverkusen, Germany), lincomycin–spectinomycin (lincomycin–spectinomycin was administered by drinking water for 5 days; the dose was 150 mg/kg chicken weight, and the withdrawal time is 7 days; Zoetis, New Jersey, USA). The control group was not treated with antibiotics. Each group was immunized with the Salmonella Enteritidis live vaccine at 2, 3, 4, and 5 days after withdrawal of the antibiotics, respectively. Meanwhile, the control group was immunized without treatment. The detailed grouping information is shown in Table 1.

Faecal samples (n = 58) were cultured in media supplemented with amoxicillin to detect beta-lactam-resistant E. coli. FecalSwab medium (0.2 mL) was inoculated into 5 mL of Luria–Bertani (LB) broth (Difco Laboratories, Detroit, MI, USA) supplemented with 10 mg/L amoxicillin (Sigma, St. Louis, MO, USA) and incubated at 37 °C overnight. The LB broth was subsequently streaked onto Chromocult Coliform Agar (Merck Millipore, Burlington, MA, USA) supplemented with 10 mg/L amoxicillin (Sigma, St. Louis, MO, USA) and incubated at 37 °C overnight. Dark blue or purple colonies were presumptively identified as E. coli, and the corresponding faecal samples were deemed positive for the presence of amoxicillin-resistant E. coli. A single E. coli colony was selected from each positive agar plate, except where morphologically distinct colonies were observed (as determined by colony and halo colour), in which case one representative isolate was selected for each colony type observed.

MCF was modified with PVOH as described above. To load antibiotics onto the internal surface of the PVOH modified MCF, individual microcapillaries of a length of PVOH modified MCF (up to 5 m) were filled using a 30G needle with freshly prepared filter sterilized antibiotic solutions of gentamicin, tetracycline, trimethoprim, ciprofloxacin, ampicillin, amoxicillin and cefotaxime (Sigma Aldrich, UK) at the following concentrations and solvents: gentamicin 5 mg mL⁻¹ in ultrapure water; tetracycline 5 mg mL⁻¹ in water; trimethoprim 3 mg mL⁻¹ in DMF; ciprofloxacin 3 mg mL⁻¹ in 1% HCl; ampicillin 10 mg mL⁻¹ in water; amoxicillin 15 mg mL⁻¹ water; and cefotaxime 4.8 mg mL⁻¹ in water. After 5 minutes of incubation, the excess solution was removed by attaching MCF lengths to a vacuum manifold and dried for 20 minutes per 1 m length, leaving behind a thin film of antibiotic as described previously.^{42 (link)} All antibiotics were loaded at concentrations that would lead to release of the breakpoint concentration into the sample (Table S-1†) based on previously determined loading efficiency quantified by LC-MS and confirmed by phenotypic AST experiments using this method.

The MICs of standard antibiotics were determined using a resazurin assay as described previously (48 (link)). Antibiotics used were amoxicillin (Sigma-Aldrich; beta-lactam antibiotic), cefuroxime (Sigma-Aldrich; beta-lactam antibiotic), moxifloxacin (Sigma-Aldrich; fluoroquinolone), and vancomycin (Sigma-Aldrich; glycopeptide), four clinically relevant antibiotics that are either commonly used to treat S. pneumoniae infections (amoxicillin, cefuroxime, and moxifloxacin) or used as a last resort, mostly in a hospital environment (vancomycin) (82 – (link)84 (link)). Briefly, a 1:2 serial dilution of the antibiotic was made in triplicates in MHL in a 96-well plate with a final volume of 100 μL. Then, 100 μL of a bacterial suspension was added to each well, except negative-control wells, to a final concentration of 5 × 10⁵ CFU/mL in 200 μL. Positive-control wells contained 200 μL of bacterial suspension (5 × 10⁵ CFU/mL) without antibiotics, and negative-control wells contained 200 μL of MHL without antibiotics or bacteria. Plates were incubated at 37°C and 5% CO₂ for 20 h before 20 μL of a 0.005% resazurin solution was added. Plates were further incubated for 90 min and fluorescence was measured (λ_em = 590 nm; λ_ex = 520 nm) using a spectrophotometer (Promega; Discover).

Faecal samples were screened for the presence of beta-lactam-resistant E. coli (specifically, resistance to penicillins and third-generation cephalosporins) by inoculating FecalSwab media (0.2 ml) into 5 ml of Luria-Bertani (LB) broth (Difco Laboratories) containing 10 mg l⁻¹ amoxicillin (a penicillin) (Sigma), and incubated overnight at 37 °C. The LB broth was then inoculated onto Chromocult Coliform Agar (Merck Millipore) supplemented with 10 mg l⁻¹ amoxicillin (Sigma) or 32 mg l⁻¹ cefoperazone (a third-generation cephalosporin) (Oxoid) and incubated overnight at 37 °C. Dark blue or purple colonies were deemed to be E. coli.