## 2 Materials and Methods
### Molecular Gene Analysis  
To study the occurrence of genes responsible for the resistance to the selected antibiotics, 42 pure strains of antibiotic-resistant bacteria were obtained by the multiple passage technique using the streak plate method. They were purified on cultivation media with antibiotics, with the incubation conditions mentioned above. The obtained microbial isolates were used as a source of DNA for further analyses. DNA was extracted using the EXTRACTME DNA BACTERIA KIT (DNAGdansk, Gdansk, Poland) and applied for PCR. In order to effectively extract the nucleic acid from the bacterial cells, microorganisms from individual isolates were first suspended in 300 uL of BacL Buffer reagent. Solutions of RNase A and proteinase K in the respective buffers (RNase Buffer and Proteinase Buffer) were prepared. An amount of 4 uL of RNase A solution was added to the above-mentioned suspension of bacteria in BacL Buffer, mixed, and incubated for 10 min at 37 \({}^{\circ}\)C. In the next step, 10 uL of Proteinase K solution was added to the sample, mixed and then incubated for another 10 min at 55 \({}^{\circ}\)C. Then, 350 uL of BacB Buffer reagent was added to the mixture and incubated for 5 min at 55 \({}^{\circ}\)C. After incubation, the sample was subjected to intense shaking for 15 s and centrifuged for 2 min at 10-12,000 rpm. The obtained supernatant was transferred to a deposit mini-column placed in a collection tube and once again centrifuged for 1 min at 10-12,000 rpm. The mini-column was placed in a new collection tube, washed with 600 uL of BacW Buffer and centrifuged for 30 s at 10-12,000 rpm. The washing was repeated using 400 uL of BacW Buffer reagent. In order to dry, the minicolumn was centrifuged for 2 min at 12-14,000 rpm, and then it was placed in a sterile Eppendorf tube. Next, 80 uL of Elution Buffer reagent heated to 70 \({}^{\circ}\)C was poured into the tube, which was then incubated for 2 min at ambient temperature. After this time, the tube was centrifuged for 1 min at 10-12,000 rpm. The isolated DNA obtained in minicolumn was stored at -20 \({}^{\circ}\)C.  
Amplification of isolated bacterial DNA was accomplished using a dedicated PCR reagent kit: REDTaq\({}^{\text{\textregistered}}\) ReadyMix\({}^{\text{\textregistered}}\) PCR Reaction Mix (Sigma-Aldrich, St. Louis, MO, USA). Product sizes were compared to DNA size markers (FastRuler Middle-Range DNA Ladder (Thermo Fisher Scientific Inc., Waltham, MA, USA)). The primers for PCR reaction were supplied by Syngen Biotech Ltd. (Wroclaw, Poland). The sequences of oligonucleotides applied as the primers for PCR reactions for individual genes are presented in Table 1.  
The PCR reaction was carried out in the following steps: initial denaturation at 95 \({}^{\circ}\)C for 3 min, 35 denaturation cycles (95 \({}^{\circ}\)C for 30 s each), connection of primers (in temperature depending on the primer, 30-60 s), extension (72 \({}^{\circ}\)C for 60 s), and the final extension (72 \({}^{\circ}\)C for 10 min). The amplification products were applied to an agarose gel immersed in Tris-Acetate-EDTA Buffer (pH 8.2-8.4) (Sigma Aldrich, St. Louis, MO, USA), along with the DNA size marker The concentration of agarose was chosen according to the size of the expected DNA product. The separation of the products by electrophoresis was carried out at 100-110 V for about 40-60 min. After electrophoresis, the gels were then immersed in ethidium bromide solution for staining. The UV light was used for the visualization of the obtained PCR products. \begin{table}
\begin{tabular}{c c c c} \hline \multirow{2}{*}{**Gene**} & **Type of the Primer** & **Sequence** & **Size (bp)** & **References** \\ \hline \multirow{3}{*}{_qnrS_} & forward & GCAAGTTCATTGAACAGGGT & \multirow{3}{*}{428} & [20] \\  & reverse & TCTAAAACGTGGTCGGCG & \\ \cline{1-1} \cline{3-4}  & forward & TCGCCGCATACACTATTCTAGAATG & \\ \cline{1-1} \cline{3-4}  & reverse & CAGCAATAAACCAGCCCAGCCGGAAG & & [21] \\ \hline \end{tabular}
\end{table} 
Table 1: Oligonucleotides used as primers for PCR reactions.