## 5 Materials and Methods
### Gene Expression Analysis  
Gene expression analysis was performed on bacterial cells adapted in BHI containing antimicrobial compounds. For the substances that reported an MBC value, sub-MBC (half of MBC) concentrations were used. Instead, for hexanoic acid and benzoic acid that did not report an MBC, a sub-MIC (half of MIC) concentration was chosen. For amoxicillin, the highest tested concentration was chosen (64 mg/L). To prepare adapted bacteria, 10\({}^{6}\) CFU/mL of an overnight _E. coli_ K88 culture were grown at 37 \({}^{\circ}\)C for 4 h after inoculation into 5 mL of fresh BHI supplemented with either 32 mg/L doxycycline, 2 mg/L colistin, 64 mg/L amoxicillin, 0.94 mM thymol, or carvacrol, 1.87 mM eugenol, 25 mM sorbic acid, benzoic acid, or hexanoic acid. Adequate controls were prepared for substances containing ethanol in their stock solutions.  
After incubation, bacterial cells were collected by centrifuging 5 min at 5000\(\times\)_g_. After supernatant removal, the pellet was resuspended in 100 \(\upmu\)L of Tris-EDTA buffer supplemented with 1 mg/mL of lysozyme and incubated at 37 \({}^{\circ}\)C for 10 min. Then, total RNA extraction from bacteria was performed using the NucleSpin RNA Kit (Macherey-Nagel GmbH & Co. KG, Duren, Germany) with DNase digestion according to manufacturer's instructions.  
RNA yield and quality were verified spectrophotometrically using A230, A260, and A280 nm measurements (\(\upmu\)Drop Plate and Varioskan LUX, Thermo Fisher Scientific Inc., Waltham, MA, USA).  
Four hundred ng of RNA were subsequently reverse-transcribed with iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to manufacturer's instruction. The cDNA obtained was used as the template for qPCR analysis, whose reactions were prepared in a final volume of 10 \(\upmu\)L, containing 5 \(\upmu\)L of 2x iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 200 (for _fueG_, _eltB_, _bsS_, _tnaA_, and _16S_) or 600 nM (_eltA_, _estA_, _estB_, _motA_, _luxS_, and _ibfB_) of each primer, 2 \(\upmu\)L of 5 ng/\(\upmu\)L cDNA, and nuclease-free water up to volume. Real-time PCR was performed using CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) under the following conditions: 3 min at 95 \({}^{\circ}\)C, followed by 40 cycles of 95 \({}^{\circ}\)C for 10 s and 60 \({}^{\circ}\)C for 30 s. The specificity of each reaction was evaluated by melting-curve analysis with 0.5 \({}^{\circ}\)C/s heating rate from 55 up to 95 \({}^{\circ}\)C.  
Primer specificity was evaluated by Sanger sequencing of control qPCR products (Microsynth AG, Balgach, Switzerland) after purification with NucleSpin Gel and PCR Clean-up Kit (Macherey-Nagel GmbH & Co. KG, Duren, Germany). All sequencing products were considered valid only if matching the expected amplicon.  
Gene expression was normalized using two reference genes, i.e., the 16S rRNA gene (_16S_) and the B subunit of the integration host factor (_ihfB_). After determining the threshold cycle (Ct) for each gene, the relative changes in gene expression of _E. coli_ K88 grown in media supplemented with treatments compared to controls were calculated using the 2\({}^{-\Delta\Delta\text{Ct}}\) method [63].  
Forward (F) and reverse (R) primers (Table 2) were designed using Primer-BLAST tool (NCBI; National Center for Biotechnology Information) and synthesized by Merck KGaA, Darmstadt, Germany.