*2.1. Chemicals and Reagents*

Tetracycline (TC, >98.0%), cephalexin (CPL, >99.0%), and sulfamethoxazole (SMX, >99.5%) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany) and selected to represent the different classes of antibiotics (tetracyclines, β-lactams, and sulfonamides, respectively) based on their frequent usage in the local livestock farms in Xinxiang City, China [28]. Tetracyclines are broad-spectrum antibiotics that inhibit bacterial protein synthesis by preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor (A) site. Resistance to tetracyclines has now emerged in many pathogenic bacteria due to genetic acquisition of *tet* genes, which include efflux genes, ribosomal protection genes, and enzymatic modification genes [29]. β-lactam antibiotics are the most widespread class of human antibacterials that inhibit bacteria by interfering with cell wall synthesis. The most major mechanism of bacterial resistance to β-lactam is the expression of β-lactamases that hydrolyze the antibiotic [30]. Sulfonamides, which are synthetic antibacterial drugs, inhibit bacterial folate biosynthesis by competing with the natural substrate *p*-amino-benzoic acid for binding to dihydropteroate synthase (DHPS), an enzyme in the folic acid synthesis pathway. Two genes, *sul*1 and *sul*2, mediated by transposons and plasmids, and expressing DHPS highly resistant to sulfonamide, have been found [31].

Methanol, acetonitrile, formic acid, and acetone of HPLC grade were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Other chemicals were of analytical grade and obtained from Yaohua Chemical Reagent Factory (Tianjin, China). Ultrapure water was supplied using a Millipore Milli-Q system (Billerica, MA, USA). Oasis HLB cartridges (hydrophilic-lipophilic balance, 6 mL, 500 mg) purchased from Waters (Milford, MA, USA) were used for the extraction and purification of the target antibiotics. Individual stock standards were prepared by dissolving antibiotics separately in methanol and were stored at −20 ◦C in brown vials.

## *2.2. Hydroponic Experimental Procedure*

A hydroponic experiment with antibiotic treatments was performed in an experimental greenhouse in the College of Life Sciences, Henan Normal University, China, during the autumn of 2016. Seeds of pakchoi (*B. chinensis* L.) were used for this study. TC, CPL, and SMX were separately added into the hydroponic solution of the test system at two concentrations: 50% of minimum inhibitory concentration (MIC) of each antibiotic, and the MIC of each antibiotic [32]. Each of the MIC values in the hydroponic solution was set as the induced dose of resistant bacteria.

Prior to testing, the seeds were surface-sterilized in 0.1% sodium hypochlorite solution for 10 min and then rinsed with sterile deionized water [33]. Seeds on a piece of sterile filter paper were placed into 10 cm sterile Petri dishes, and 10 mL of sterile water was added. Then, the Petri dishes were covered with their lids and maintained in a dark incubator at 25 ± 2 ◦C. After germination, the seeds were transferred to a plastic cuboid hydroponic tank (45 × 20 × 17 cm). All tanks were wiped with 75% ethanol and thoroughly rinsed with deionized water before first use. After that, each tank was filled with 12 L of Hoagland nutrient solution [34] or Hoagland nutrient solution supplemented with an antibiotic. The treatments were as follows: (1) control with no antibiotics added; (2) TC-treated (at concentrations of 8 and 16 mg·L−1, respectively); (3) CPL-treated (at concentrations of 32 and 64 mg·L−1, respectively); and (4) SMX-treated (at concentrations of 38 and 76 mg·L−1, respectively). Each treatment was designed with three replicates, and a total of 21 hydroponic tanks were used in the present study. Additionally, each tank had nine cylindrical holes (4 cm depth and 3 cm diameter) in its cover containing sponges (3 × 3 × 2.5 cm) in individual cylinders as a rooting medium. Five uniform seeds were planted per hole and irrigated with half-strength Hoagland solution every 2 days until the roots of the seedlings were immersed in the solution. Finally, only one or two strong seedlings were selected to leave in each hole, and they were grown directly in nutrient solution.

During the experiment the room conditions were maintained at 25 ± 2 ◦C in daylight and 18 ± 2 ◦C at night, with a relative humidity between 65% and 70%. Each planter was equipped with an electric aeration pump and was aerated for 2 h every day. Because of evapotranspiration from the vessels, lost water was supplemented with fresh nutrient solution without the addition of extra antibiotics. Pakchoi was harvested after 55 days of cultivation. Then, the sponges attached to the vegetables were trimmed off. The plants were rinsed first with tap water and then with deionized water and dried on adsorbent paper. The growth parameters and abundance of endophytic bacteria were measured immediately. Antibiotic analyses and DNA extraction from endophytic bacteria were completed within two weeks of sampling.
