E coli dh5a

The fluoroquinolone-resistant and ESBL-producing S. Derby isolates were inoculated into 200 mL of Luria-Bertani (LB) broth and cultivated for 12 h with shaking of 150 rpm. Then, the plasmids in 200 mL of broth were extracted by a large-volume preparation method using commercial EasyPure Hipure Plasmid Maxiprep Kit (Transgen, Beijing, China). Plasmid profiles were obtained for each isolate using electrophoresis. A PCR-based plasmid typing method was performed to trace the drug-conferring plasmids using 18 pairs of primers representing FIA, FIB, FIC, HI1, HI2, I1-Ig, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA plasmid replicons [16 (link)].
Plasmid transformation into E. coli DH5a (TaKaRa Biotechnology, Dalian, China) was conducted for the extracted plasmids to assess β-lactam resistance dissemmination by plasmid. Transformants were selected on LB agar containing 50 μg/mL ampicillin. The highly resistant S. Derby isolates were also tested for resistance transmission ability among isolates through conjugation experiment with rifampin-resistant E. coli c600 as recipients according to previous literature [17 (link)]. Transconjugants were selected on MacConkey agar plates containing ceftriaxone (16 μg/mL) and rifampin (50 μg/mL).

The PLE ORF was cloned by the T-A cloning technique. Total RNA was extracted. The cDNA synthesis by RT-PCR was performed using M-MLV (TAKARA, DaLian, China) with an oligo (dT) primer(TAKARA, DaLian, China) following the protocol provided with the kit. RT-PCR products were used as templates for the amplification of the complete PLE ORF by two gene-specific primers according to the sequence of PLE cDNA (X63323):
PLE-F:5′-ATGTGGCTTCTCCCGCTGGTCCTGA-3′
PLE-R:5′-TCACTTTATCTTGGGTGGCTTCTTT-3′
The PCR products were detected and separated by 1% agarose gel electrophoresis and then connected to the pMD18-T vector (TaKaRa, DaLian, China). The ligation products were transformed into E. coli DH5a (TaKaRa, DaLian, China) and at least 20 positive clones from each pig were sequenced by Tsingke (Wuhan, China). A total of over 800 clones were sequenced from the livers of 24 pigs.
All the sequences of PLE ORF were translated into AA sequences by Primer Premier 5.0. The translated AA sequences were aligned by the ClustalX2 software and then classified into PLE A to PLE G according to the 25 variable AA sites, then the mRNA abundance of each PLE isoenzyme in the liver of the four age groups and two breeds was calculated.

Conjugation experiments were conducted in bla_CTX-M-positive strains by liquid mating-out assay in LB-medium using a sodium azide-resistant E. coli J53 as the recipient. Transconjugants were selected on MacConkey agar containing cefotaxime (2 mg/L) and sodium azide (300 mg/L). For transformation experiments, plasmid DNA from the bla_CTX-M-positive strains were extracted using Qiagen Plasmid Midi Kit according to the manufacturer’s instructions (Qiagen, Germany). Purified plasmids were transformed into E. coli DH5a (TaKaRa Biotechnology, Dalian, China). Selection of transformants was performed on MacConkey agar containing 2 mg/L cefotaxime. MICs and the presence of bla_CTX-M gene of transconjugants and transformants were determined as described above.