## 4 Materials and Methods
### RNA Isolation and Quantitative Real-Time RT-PCR  
To isolate the RNA of the EDL933 strain used in the real-time RT-PCR experiments, bacteria were inoculated in 8 mL of modM9 at the initial concentration of 1 \(\times\) 10\({}^{6}\) CFU/mL in the presence or in the absence of peptides, which were used at sub-lethal concentrations of 2 and 16 uM for Esc(1-21) and Esc(1-18), respectively. Cultures were incubated at 28 \({}^{\circ}\)C for 24 h with 250 rpm agitation and stabilized with RNA Protect Bacteria Reagent (Qiagen). RNA extraction was performed using a Presto Mini RNA Bacteria kit (Geneaid, New Taipei City, Taiwan), DNA contamination was eliminated using DNase I (Epicentre, Singapore) for 20 min at 37 \({}^{\circ}\)C, and RNA was precipitated with 0.7% isopropanol and 0.3 M sodium acetate. The absence of residual DNA was verified through PCR using specific primers. The quantity and the integrity of RNA were determined using a UV-VIS one-drop micro-volume spectrophotometer (DeNovix-Resnova, Wilmington, DE, USA) at 260 nm. RT-PCR was used to investigate the transcription levels of genes involved in the regulation of biofilm formation and dispersion (_csr_A, _hua_, _uri_B), in flagellar synthesis (_flh_C, _flh_D, _fli_C), in the stringent response (_rel_A, _spo_T), and in oxidative (_sod_C, _kat_E) and osmotic stress responses (_osm_B). To perform RT-PCR, an SYBR Green kit (Luna Universal One-Step RT-qPCR, BioLabs, Cambridge, MA, USA) was used. The PCR cycling conditions were as follows: cDNA synthesis at 55 \({}^{\circ}\)C for 10 min, a denaturation program at 95 \({}^{\circ}\)C for 1 min, and amplification and quantification were repeated 40 times: 95 \({}^{\circ}\)C for 10 s and 60 \({}^{\circ}\)C for 30 s. The specificity of PCR was determined with melting curve analyses (55 \({}^{\circ}\)C to 95 \({}^{\circ}\)C with a heating rate of 0.3 \({}^{\circ}\)C/s). Forward and reverse primers for the analyzed genes were designed using the Pel Primer software (Table 2).  
\begin{table}
\begin{tabular}{c c c} \hline
**Oligo Name** & \multicolumn{2}{c}{**Sequence 5\({}^{\prime}\)-3\({}^{\prime}\)**} \\ \hline \multirow{2}{*}{_16s_} & For & Rev \\  & CATCCACAGAACTTTCCAGAG & CCAACATTTCACAACCAG \\ \hline \multirow{2}{*}{_csr_A} & For & Rev \\  & TTAGTAACTGGACTGCCTGGG & GTTGGAGACCCTCATGAT \\ \hline \multirow{2}{*}{_flh_C} & For & Rev \\  & CGACTGGTTTATGCTG & CTGGTGAGGTTGGGTAATAA \\ \hline \multirow{2}{*}{_flh_D} & For & Rev \\  & CCGAGTTGCTGAAAACACATTTA & ATTATGCCGAGACGAACGA \\ \hline \multirow{2}{*}{_fli_C} & For & Rev \\  & TTGCCGACTATACAGTCTCTTAC & TTGGTAGTGGTTTGTTTCAG \\ \hline \multirow{2}{*}{_Hha_} & For & Rev \\  & AGGAAAGGATTCTTGTCGGTACAG & TCGTTGCCAGACAATTGCAC \\ \hline \multirow{2}{*}{_kat_E} & For & Rev \\  & GATTCATACCTCCGCCG & GTCTCTTCATCCAAAACCAG \\ \hline \multirow{2}{*}{_nir_B} & For & Rev \\  & TTACCTCGACGAAAGCAAAG & GCAGTTTTACAACACCGATAGA \\ \hline \multirow{2}{*}{_osm_B} & For & Rev \\  & GTTCTAACGTGCTAAAACGGG & CCTAATGTACCAAACGTAC \\ \hline \multirow{2}{*}{_relA_} & For & Rev \\  & GATTACTGCTTCCGTTATCC & CGACCATACACTTCAGCTTTA \\ \hline \multirow{2}{*}{_sod_C} & For & Rev \\  & GTCGAGATGAAACCTGCTGAG & TCCAGACACCTTATCGGTTTTCA \\ \hline \multirow{2}{*}{_spo_T} & For & Rev \\  & ATGCTGTGATGGAATA & TCTTTCCTTCTTCCGATATCAAACT \\ \hline \end{tabular}
\end{table}
Table 2: Primers used in this study for reverse transcription–quantitative PCR.  
To determine the efficiency of each primer pair, a series of five ten-fold dilutions were performed, and standard curves were generated. R2 values or correlation coefficients >0.95 were considered optimal correlations between values. The 16s rRNA, a housekeeping gene, was used to normalize the levels of target gene expression detected between treated and untreated strains by measuring the changes in fold expression using the 2-\(\Delta\Delta\)CT method [53]. Bacteria without treatments were used as a calibrator sample (control sample). All RT-PCR experiments were conducted at least in triplicate.