*2.7. Screening for Mutations*

The genes *ramR*, *marR*, *soxR* and *rpsJ* were amplified and sequenced with the primers described previously [18,21].

Standard PCR protocols and conditions were used in the following way: initial denaturation at 94 ◦C for 5 min; 35 cycles of 94 ◦C for 30 s, 52 ◦C for 1 min 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, Germany).

Sequencing was performed with the Mix2Seq Kit (Eurofins Genomics).

Sequence analysis was performed with Serial Cloner v2.6 and BLAST (Basic Local Alignment Search Tool, https://blast.ncbi.nlm.nih.gov/Blast.cgi).

#### **3. Results**

Two *Klebsiella pneumoniae* isolates (MurTR-KL001 and MurTR-KL002) were randomly sampled from the river Mur during a study not linked to tigecycline.
