Plasmid PCR-based replicon typing (PBRT) was performed on the 39 blaCTX-M/blaCMY-2-positive selected isolates for identification of 21 replicon plasmids as previously described [34 (link), 35 (link)]. Plasmids were purified in wild type isolates using the QiaFilter Midi kit (Qiagen Inc.), following the manufacturer’s instructions. Purified plasmid DNA of 30 ESBL/AmpC-producer isolates randomly selected (16 blaCMY-2-positive and 14 blaCTX-M-positive isolates, some of which also carried the blaTEM gene) was electroporated into E. coli DH10B Electromax competent cells (Invitrogen, Calsbad, CA).
Transformants were selected on Mueller Hinton agar supplemented with ceftriaxone 2 μg/ml [36 (link)]. Up to five transformants, when available, were screened by PCR for the presence of incompatibility plasmids and of all AMR and virulence genes present in the corresponding wild type strains. Transformants carrying ESBL/AmpC genes were subsequently tested for their susceptibility to the 14 antimicrobials as mentioned above.
The methodological approach that summarizes the main steps of this study can be found inS1 Fig .
Transformants were selected on Mueller Hinton agar supplemented with ceftriaxone 2 μg/ml [36 (link)]. Up to five transformants, when available, were screened by PCR for the presence of incompatibility plasmids and of all AMR and virulence genes present in the corresponding wild type strains. Transformants carrying ESBL/AmpC genes were subsequently tested for their susceptibility to the 14 antimicrobials as mentioned above.
The methodological approach that summarizes the main steps of this study can be found in
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