Susceptibility to antimicrobial agents and heavy metals The isolates were measured for in vitro susceptibility
to click here antimicrobial agents according to the guidance of the Performance Standards for Antimicrobial Disk Susceptibility Tests of the Clinical and Laboratory Standards Institute (CLSI) (2006, Approved Standard-Ninth Edition, M2-A9, Vol. 26 No.1). Mueller-Hinton agar medium (Oxoid, UK), and the discs (Oxoid, UK) were used in this study. Examined antimicrobial agents included: 10 μg ampicillin (AMP), 30 μg chloramphenicol (CHL), 10 μg streptomycin (STR), 10 μg gentamicin (CN), 30 μg kanamycin (KAN), 5 μg rifampicin (RIF), 100 μg spectinomycin (SPT), 30 μg tetracycline (TET), 5 μg trimethoprim (TM), and 25 μg SXT (sulfamethoxazole (23.75 μg)-trimethoprim
(1.25 μg). The assays were performed in triplicate experiments, and reference strain Escherichia coli ATCC25922 was purchased from the Institute of Industrial Microbiology (Shanghai, China) and used for quality control. Broth Dilution Testing (microdilution) was used to measure quantitatively the minimal inhibitory concentration (MIC) in vitro of the tested antimicrobial agents against the stains, according to the Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically (2006, CLSI, Approved Standard-Seventh Edition, M7-A7, Vol.26 No.2). Similarly, the MICs of the heavy metals, including Hg(NO3)2, Cd(NO3)2, Pb(NO3)2 and ZnCl2 (Sigma-Aldrich, USA), as well as CuSO4 (Songong, Endocrinology inhibitor China), were also determined. Conjugation Conjugation experiments were performed using the strains with appropriate selective markers as the donors (Table 1) and a chloramphenicol-resistant stain of E. coli (stain MG1655, a gift from Dr. Liping Zhao) as the recipient, according to the method described by Waldor et al.  with slight modification. The antimicrobial agents used for selection in plate mating assays included: chloramphenicol (30 μg/ml), sulfamethoxazole (128–160 μg/ml), streptomycin
(30–60 μg/ml). Briefly, recipient and donor strains were individually Thiamine-diphosphate kinase cultured to Alpelisib chemical structure log-phase, the latter was treated with mitomycin C (50 ng/ml) for 1 h at 37°C to increase transfer frequency of SXT elements (Beaber et al., ). Cell cultures were harvested by centrifugation, and mixed at a ratio of approximately 1:1. The cell mixture was resuspended in 0.2 ml LB, and then spotted onto LB agar plates. Mating was performed at 37°C for 6 h. Cells from the mating plates were harvested in 200 μl LB broth, and serial dilutions were spread onto the appropriate selective agar plates. The successful transfer of ICEs into the recipient strain was confirmed by colony PCR using the primers for characterizing the ICEs in this study (Table 2). The transfer frequency was calculated as the number of tansconjugants in mating cell mixture per donor cell.