## 2 Materials and Methods
### Detection of Genes Encoding Resistance to Antimicrobial Substances and Virulence Factors  
Using simplex and multiplex PCR, genes encoding resistance to antimicrobial agents and virulence factors were found. The individual reaction PCR protocols can be summed up as follows. Following an initial denaturation at 94 \({}^{\circ}\)C/95 \({}^{\circ}\)C for 1 to 12 min, there were 25-34 cycles with the following parameters: denaturation for 30-60 s at 94 \({}^{\circ}\)C/95 \({}^{\circ}\)C, annealing for 30-60 s at 52-65 \({}^{\circ}\)C (based on primer sequence), and extension for 25 s-3 min at 68 \({}^{\circ}\)C/72 \({}^{\circ}\)C (dependent on the length of the amplified section). The last phase was to add a final extension step for two to ten minutes at 72 \({}^{\circ}\)C. The steps outlined in Section 2.2 were followed for the composition of the reaction mixture, the PCR reaction duration, and the visualization of the DNA fragments.  
Table 1 contains a list of all primers used in each PCR reaction.  
The presence of genes encoding quinolone resistance--_qnr_A, _qnr_B and _qnr_S--and tetracycline resistance--_tet_A and _tet_B--was investigated. Furthermore, the prevalence of genes encoding mobile genetic elements (MGEs)--integons (_Int1_ and _Int2_) and transposons (_Tn3_)--was assessed. These MGEs have the ability to capture and express genes in gene cassettes, thereby significantly increasing antimicrobial resistance in Gram-negative bacteria [40].  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Gene** & **Primer Sequences (5\({}^{\circ}\)–3\({}^{\circ}\))** & **Annealing** & **Product Size** & **References** \\ \hline _16S rRNA_ & GACCTGGTTTAGTTCACAGA & 55 \({}^{\circ}\)C & 585 bp & [22] \\  & CACACGCCTGAGCTGACCA & & & \\ \hline _chu_A & ATGATCATCCGCCGCGCGCTG & & & \\  & AAACGCGCTCCGCCAAT & 65 \({}^{\circ}\)C & 281 bp & [29] \\ _yi_A & TGTTCGCGATCTTGAAGCAAACGT & 65 \({}^{\circ}\)C & 216 bp & [29] \\  & ACCTGGACAAACGCCTG & & & \\  & GGCGGCGAGACAGAACGCG & 65 \({}^{\circ}\)C & 152 bp & [29] \\  & TTGCTGAGCTTTGCAGACCCG & & & \\ \hline _qnr_A & ATTCTCACGCAGGATTTG & & & \\  & GATCCGCAAAGGTTAGGTCA & & & \\  & GATCGCAAAGCCAGAAGG & & & \\  & AGATGCCGGTGTGTGTCC & & & \\  & ACGACATTGTGTGCAATCTCTGCAA & & & \\  & TAAAATTGGCACCCCTGAGCC & & & \\  & tetA & GGCCTGATGCCG & & & \\  & AAGCAGCAGATTCAG & & & \\  & GAGAGCAGATTCAG & & & \\  & TTTAGTGCTATTCTTCCC & & & \\ \hline _Int_1 & GGGTCAAGGATTCG & & & \\  & ACATGCGTTGAATTCATCTG & & & \\  & ACATGCGTTGAATTCATCTG & & & \\  & CACGGGGATATGCGAGAAAAGGT & & & \\  & GATGAGAGGTCAGAAAATG & & & \\  & CATGAGAGGTCCGACACAGG & & & \\  & ACCACCACTCGGCACCACACTG & & & \\ \hline _lpsMT_ II & GCCATTGTGTGTATACTGTTG & 63 \({}^{\circ}\)C & 272 bp & [36] \\  & CATCCGAGAGATGATGATGAGAC & & & \\  & GCGAGATTCTTCTGCTGCTGCTG & & & \\  & TTGGAATTACAACTTTGAGGTCCATC & & & \\  & GACGCGCTGTACTGCAGGGTGTGGCG & & & \\  & ATATCTTCTTCCGAGGGTGTCAATA & & & \\  & GCGCGACACATTAGGAGG & & & \\  & GCTGGGAAACGGGATGATG & & & \\  & CACACACAAAACGGGGACCTG & & \\  & CTCCGCACATGCTTCCT & & & \\ \hline \hline \end{tabular} Description: _16S rRNA_ = _E: coli_ identification_, _chuxt_, _yiA_, _TspE4.C2_ = phylogenetic grouping; _qnr_A_, _qnr_B_, _qnr_S = quinolone resistance; _tetA_, _tetB_ = resistance to tetracycline; _Int1_, _Int2_ = integron; _Tn3_ = transposon; _lpsMT II_ = capsule; _finA_ = type I fimbie; _pnpC_ = P fimbie; _inA_ = receptors for aerobactin; _cnC_ = colline V.  
\end{table}
Table 1: List of primers used for detection of _E. coli_, phylogenetic analysis, and detection of genes of antimicrobial resistance and virulence factors.  
Moreover, we evaluated the presence of genes encoding the following virulence factors: capsule (_kpsMT_ II), type 1 fimbiae (_fim_A), P-fimbriae (_pap_C), receptors for aerobactin (_iut_A), and colicine V (_cva_C).