Microbiological procedure
LYSED
Samples were centrifuged for 10 min at 9800 rpm, following which the supernatant was discarded. The bacterial pellet was then suspended in 20 mM Tris–HCl, pH: 8.0; 2 mM EDTA; 1.2% Triton (Triton X-100) and cells lysed with 20 mg/ml lysozyme, followed by overnight incubation in a water bath at 37 °C. Finally, manufacturer’s instructions were followed for DNA extraction procedure using QIAamp Mini DNA kit (Qiagen, Hilden, Germany), and the extracted DNA was then stored at −20 °C until further use.ABI StepOnePlus Real-Time PCR system (Applied Biosystems, USA) was used to perform the reaction in Microamp fast optical 96-well reaction plates (Applied Biosystems, Foster City, CA, USA) covered with optical adhesive film (Applied Biosystems). The cycle conditions were: 50 °C /2 min; 95 °C /10 min; 50 cycles of 95 °C /15 s and 58 °C /1 min. The oligonucleotide primers used in the study are listed in Supplementary Table After the amplification of the extracted DNA samples, the obtained Ct values were plotted against the standard curves, and the absolute number of 