The cultures were centrifuged at 8,000 × g to precipitate bacterial cells. Total RNA was extracted using the hot phenol method as previously described with modifications (11 (link)). Subsequently, the bacterial cells were washed two times with RNAse-free saline or phosphate-buffered saline (PBS; cat. no. E607016-0500; BBI solutions, Cardiff, UK). Then, 400–600 µl TES solution was added according to the precipitation amount, and the bacterial cells were resuspended. The same amount of phenol-water (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was added followed by violent mixing. Centrifuge tubes containing a mixture of each sample, TES, and phenol-water were agitated at 65°C for 30–60 min in a Thermomixer Compact 5350 (Eppendorf, Hamburg, Germany), and then, the tubes were placed on ice and allowed to stand for 5 min. Then, the mixtures were centrifuged at 11,000 × g for 10 min at 4°C. The upper aqueous phase was selected and transferred to a new tube. Subsequently, a 1/2 volume of TRK-1002 lysis-solution and 2/3 volume of 95% ethyl alcohol was added to the upper aqueous phase, followed by vortex blending. Total RNA was then extracted using a TRK-1002 Purification kit (LC Sciences, Houston TX, USA), following the manufacturer's instructions. RNA quality was evaluated using an Agilent Bioanalyser (Agilent Technologies, Inc., Santa Clara, CA, USA).