2.3. Bacterial Identification

By amplification and sequencing of a fragment of 16S rDNA, bacterial isolates grown on inoculated plates from skin lesions, MK, and HK were classified to the species level. Complete genomic DNA was extracted from bacteria using the Thermo Scientific Gene JET Genomic DNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA). Afterwards, this fragment was amplified using the universal primers SD-Bact-0008-a-S20 (5′ AGA GTT TGA TCC TGG CTC AG 3′) and SD-Bact-1492-a-A-19 (5′ GGT TAC CTT GTT ACG ACT T) [14]. Polymerase chain reactions (PCR (were carried out in a 50 μL reaction mixture that included 5 pmol of each primer, 0.2 mM dNTPs mix, 10X DreamTaq Buffer, 2 mM MgCl₂, 1.25 U DreamTaq DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) and 1 μL of colony DNA (~ 100 ng/ul). The PCR profile was as follows: 2 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 40 s at 52 °C and 1.3 min at 72 °C and a final step 5 min at 72 °C. The products of the polymerase chain reaction were electrophoresed on a 1% agarose gel and visualized using ultraviolet transillumination. Before sequencing the PCR product, an ExoSAP-IT purification step was performed. For this, 9 µL of each PCR product were mixed with 0.5 µL of ExoI (20 U µL⁻¹) (Thermo Fisher Scientific, Germany). The PCR products were enzymatically purified (37 °C for 15 min, 80 °C for 15 min). Subsequently, they were then sequenced using Sanger technology at Macrogen Spain (Madrid, Spain). Using the Basic Local Alignment Search Tool (BLAST) software, the sequences were then compared to those in the GenBank databases (www.ncbi.nlm.nih.gov/blast, accessed on 27 April 2021).