*2.3. Detection of Insertion Sequences*

DNA extracts were examined for the detection of different insertion sequences associated with ESBL genes, performing PCRs assays using the specific primers and conditions showed in Table 3 [27,32,33].

The PCRs were performed in a final volume of 25 μL containing 2 μL of DNA extract mixed with 2.5 μL of 10× buffer (Bioline, London, UK), 5 μL of dNTPs (Bioline, London, UK), 1.5 μL of MgCl2 50 mM (Bioline, London, UK), 2 μL of each primer (Sigma-Aldrich, Madrid, Spain), and 1.5 U of Inmolase™ DNA polymerase (Bioline, London, UK), in a DNA thermal cycler GeneAmp® PCR system 2700 (Applied Biosystems Division, Foster City, CA, USA). Amplification conditions were modified in order to improve the specificity using an initial denaturation at 94 ◦C for 12 min, followed by 35 cycles of DNA denaturation at 94 ◦C for 1 min, and primer annealing temperature depending on the IS (Table 3), primer extension at 72 ◦C for 2 min, and a final elongation at 72 ◦C for 10 min. PCR products were separated by electrophoresis on 1% agarose gels and were visualized under UV light after staining with ethidium bromide.


**Table 3.** Primers and conditions used for the amplification of insertion sequences.

<sup>1</sup> Fw: forward; <sup>2</sup> Rv: reverse; <sup>3</sup> T (◦C): annealing temperature.
