*2.2. DNA Extraction and Detection of Integrons*

DNA extraction was performed with DNeasy® Blood & Tissue kit (Qiagen, Barcelona, Spain), using a pre-treatment protocol for Gram-negative bacteria, and following the manufacturer's instruction. The quantity and quality of the DNA was analyzed using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).

Detection of class 1, class 2, and class 3 integrons in ESBL-producing *E. coli* was performed according to PCR, as described by Mazel et al. [31], and using only the primers shown in Table 2.


**Table 2.** Primers used for the detection of integrons.

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

DNA amplification was performed in a DNA thermal cycler GeneAmp® PCR system 2700 (Applied Biosystems Division, Foster City, CA, USA) 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, Steinheim, Germany), and 1.5 U of Inmolase™ DNA polymerase (Bioline, London, UK). The conditions of the amplification were as follows: initial denaturation at 94 ◦C for 10 min, followed by 30 cycles of DNA denaturation at 94 ◦C for 45 s, primer annealing at 62 ◦C (*intI1* and *intI2*) or 60 ◦C (*intI3*) for 35 s, primer extension at 72 ◦C for 2 min, and a final elongation at 72 ◦C for 7 min. Positive and negative controls [17] were included in all PCR assays, and 1 kb ladder (Invitrogen) was used as a molecular size standard. After amplification, PCR products were separated by electrophoresis on 1% agarose gel in 1× TBE buffer, stained with ethidium bromide and visualized by UV transillumination. *E. coli* C828, *K. pneumoniae* C933 (provided both by Centro de Investigación Biomédica de la Rioja) and *E. coli* isolated from hospital inpatients, confirmed as carrying *intI2* by DNA sequencing, were used as positive controls for *intI1*, *intI3*, and *intI2*, respectively.
