*2.4. Plasmid Replicon Typing*

Identification of replicon types of the 18 major plasmid incompatibility (Inc) groups present in *Enterobacteriaceae* was performed by multiplex PCR.

Standard PCR protocols and conditions were used in the following way: initial denaturation at 94 ◦C for 5 min; 30 cycles of 94 ◦C for 1 min, 60 ◦C for 30 s and 72 ◦C for 1 min; and a final incubation for 5 min at 72 ◦C. We used Taq DNA polymerase and dNTPs from QIAGEN (Hilden, Germany), and a T3000 Biometra thermocycler (Biometra, Gottingen, Germany).

The protocol allows detection of the following Inc groups: Hl1, Hl2, I1-Iγ, X, L/M, N, FIA, FIB, W, Y, P, FIC, A/C, T, FIIs, F, K, B/O [27].

#### *2.5. Transformation by Electroporation*

Preparation of Plasmid-DNA was performed with the QIAprep Spin Miniprep Kit (250) (QIAGEN).

Plasmid-DNA was desalted before electroporation, and therefore 2–3 μL of plasmid-DNA were transferred on a MFTM Membrane Filter (0.025 μm VSWP, Merck), which was placed on the surface of double distilled water. Dialysis was performed for about 15 min.

Competent cells were made with two overnight cultures (each 50 mL, OD of 0.4), which were incubated on ice for 25 min, therefore reaction tubes were cooled in advance, followed by centrifugation for 10 min at 4 ◦C and 4.000 rpm (Eppendorf, Centrifuge 5810R). After decantation, pellets were re-suspended in 100 mL ice-cold glycerine solution (10%). After repeating this step, an additional washing step was performed, and the two pellets together were re-suspended in 10 mL glycerine solution (10%). A last washing step and resuspension were performed with 1 mL glycerine solution (10%). Aliquots of 50 μL were prepared and stored at −20 ◦C.

Electroporation was performed with 2 μL Plasmid-DNA and 40 μL of competent cells. Reaction tubes were cooled in advance and the DNA-cell suspension was incubated on ice for 5 min. Subsequently, the cell suspension was transferred into a sterile electro-cuvette, and transformation was performed at 2500 V using the electroporator (Eppendorf Eporator®). After the transformation, 400 μL of fresh LB liquid media were added and the cell suspension was re-transferred into the reaction tube. Incubation was performed for 40 min at 37 ◦C. Afterwards 100 μL of the cell suspension were plated on selection LB (lysogeny broth) plates (tetracycline 3 μg/mL or tigecycline 1 μg/mL) and a final incubation was performed over night at 37 ◦C.
