Braenderup isolates were characterized. Plasmid DNA was purified from resistant wild-type

isolates by the alkaline lysis method [42] and then transformed into the competent E. coli strain pir116 (STRR), which was prepared by the CaCl2 method. Transformants were selectively grown on LB agar plates supplemented with AMP (100 μg/ml) and further tested for resistance to CHL, TET, and KAN, but not for resistance to STR, since the recipient strain was inherently resistant to streptomycin. The antibiotic resistance genes bla TEM, aadA, and bla CMY-2, class 1 integron as well as the insertion sequence IS26 and its related DNA fragments were amplified using the primers listed in Table 4. The genes bla SHV and bla CTX-M3 and M14 were also detected by the multiplex method [43]. The R-plasmids of each transformant Osimertinib were purified by use of the Geneaid Plasmid Midi Kit (Geneaid, Taiwan) and were digested with HindIII (New England Biolabs, USA) to determine similarity. Plasmid DNA fragments were separated by electrophoresis through a 0.6 %

SeaKem GTG agarose gel (Cambrex Bio Science Rockland, Inc., Rockland, ME, USA) at 25 V for 16 h. The Small molecule library cost PCR product of class 1 integron was purified by DNA Clean/Extraction kit (GeneMark, Taiwan) and sequenced by Mission Biotech co. (Taiwan). Table 4 The PCR primers for PCR and size of PCR products Primer Target DNA sequence (5′ to 3′) Product Sizesize Note Tem-F bla TEM GAAGATCAGTTGGGTGCACGAGT 550 bp This study Tem-R   CAACTTTATCCGCCTCCATCCAGT     STR-F1 aadA2 AGACGCTCCGCGCTATAGAAGT 203 bp (46) STR-R1   CGGACCTACCAAGGCAACGCT     CS-F Clostridium perfringens alpha toxin CS region GGCATCCAAGCAGCAAG Variable (47) CS-R   AAGCAGACTTGACCTGA     1.9CS-F Flanking region of CS region CTGCTGCGTAACATCGTTGCT Variable This study 1.9CS-R   GGCGAGATCATCAAGTCAGT     ColE1-F ColE1

oriT CAAATGCTGTCCTTCCAGTGT 225 bp This study ColE1-R   CTCAGTTCGGTGTAGGTCGT     F-F IncFI oriT CAACAACGCGCCGACACCGT 288 bp This study F-R   CCCTTCCTGTCGACGCTTCT     R100-F IncF2 oriT CCACCAAAAGCACCACACACT 266 bp This study R100-R   AGACACTCCTAGCAGCGCCT     pSC138-F IncI oriT TGTCACGAACATCTGCCAGT 193 bp This study pSC138-R   GAGAGAAAGTGCCCATGGCT     IS26in-F IS26 GGCACTGTTGCAAAGTTAGC 820 bp DQ390455.1 IS26in-R   GGCACTGTTGCAAATAGTCG     IS26out-F Variable GCTAACTTTGCAACAGTGCC Variable DQ390455.1 IS26out-R   CGACTATTTGCAACAGTGCC     Tn-F Tn ACCTAGATTCTACGTCAGTAC Variable (35) AmpC-F AmpC CAAGTTTGATTCCTTGGACTCT   AY253913 AmpC-R   CTCATCGTCAGTTATTGCAGCT     SugE-R sugE GCCTGATATGTCCTGGATCGT     Plasmid conjugation and incompatibility group Transferability of R plasmids from each RFLP group was determined by performing the conjugation test following a previously described method [44] with NAL-resistant S. Typhimurium LBNP4417 as the recipient strain. Briefly, 0.6 ml of overnight culture of donor strain was mixed with 1 ml of the overnight recipient strain.