Isolates
The rectal swabs collected on each visit from the target animals were pooled and tested as one analytical sample. A total of 1230 E.coli isolates were used in the study, which including 858 isolates identified from rectal swabs of pigs in different province in China by using the biochemical identification and PCR method according to ‘Bergey’s Manual of Determinative Bacteriology’ [36]: Heilongjiang (n=293), Jilin (n=151), Liaoning (n=238), Henan (n=97), Shandong (n=30), Hubei (n=20), and Yunnan (n=29) from June 2014 to April 2017, and 372 E.coli strains were respectively donated by National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (n=108), Husbandry and Veterinary College, Jilin University (n=112), and College of Animal Husbandry and Veterinary Science, Henan Agricultural University (n=152). All of the bacterial isolates were confirmed by polymerase chain reaction (PCR) [37].
Chemicals and reagents
Pure powder of APR was purchased from Zhejiang Hisun Pharmaceutical Co., Ltd., Taizhou, China. MacConkey medium, eosin-methylene blue medium, Mueller-Hinton (MH) broth, and MH agar were supplied form Qingdao Hope Bio-Technology Co., Ltd., Qingdao, China. Premix TaqTM Version 2.0 plus dye and DL1000 DNA Marker were obtained from Takara Biotechnology Co., Dalian, China. All primers used in the study were synthesized by the Sangon Biotech Co., Ltd., Shanghai, China.
Antibacterial susceptibility testing
Broth microdilution testing was performed according to the CLSI document M07-A9 [38] at the following laboratories: Department of Microbiology, Department of Pharmacology and Toxicology, and Pharmacy Department in Northeast Agricultural University, Harbin, China. APR stock solution of 5120 µg/mL was prepared. Working solutions in plates were prepared by two-fold serial dilutions in MH broth. Finally, each well of 96 well plates contains approximately 5 × 105 CFU/mL E.coli and APR concentrations ranged from 0.5 to 256 µg/mL. Plates were placed in a constant temperature incubator at 37°C for 20 h. Quality control (QC) isolate E.coli ATCC 25922 (purchased from the NATIONAL CENTER FOR MEDICAL CULTURE COLLECTIONS, Beijing, China) was used on each day of testing as recommended by CLSI [38]. Only those results, for which the QC MICs were within the established reference range (4-8µg/mL), were used in the study [39]. All MICs determinations were performed in triplicate.
Data analysis
The definitions of the subsets, lognormal distribution, skewness, kurtosis, and COWT are presented in Table 1. The MICs were transformed into log2 values in order to analyze the MIC distributions. The kurtosis and skewness of each MIC distribution were tested. To confirm the presence of more than one MIC distribution, frequency distributions of MIC data were analyzed by nonlinear least squares regression analysis based on the following Cumulative Gaussian Counts equation: Z=((X-Mean))⁄SD, Y=N*zdist(z) according to the previous study [40], in which the Mean is the average of the original distribution, from which the frequency distribution was created; SD is the standard deviation of the original distribution (calculated by Graphpad prism 6.0 software, San Diego, CA). Three parameters, the total number (N) in the presumed unimodal distribution, the mean, and SD (both log2) were estimated. N was estimated rather than taken as a constant in the regression, because of the desire to fit the data to the distribution without assuming that N truly contained only wild-type isolates [22]. The NORMINV and NORDIST functions in Microsoft Excel were used to set the WT distribution cutoffs which were used to determine the MIC that encompass at least 95% of that distribution [22, 41].
Molecular characterisation of mechanisms of resistance to APR
A total number of 310 E.coli strains from different MIC subsets (0.5-256 µg/mL) were conveniently selected for the detection of resistance genes in E. coli that confer resistance to APR by PCR. The primers used in this study are presented in Table 2. Genomic DNA was extracted using a TIANamp Bacteria DNA Kit (TIANGEN BIOTECH (BEIJING) CO., LTD.) according to the manufacturer’s instructions. Then, 2µL (400 ng/µL) was added to a reaction mixture containing 25 µL Premix TaqTM Version 2.0 plus dye, 13µL sterile ddH2O, 5µL 10µM primer F and 5µL 10µM primer R. Amplification conditions were 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s (52 °C for apmA) and 72 °C for 1 min, and a final elongation at 72 °C for 10 min. PCR products were analysed on 1.5% (w/v) agarose gelsstained with ethidium bromide. The amplified products were sequenced by the Sangon Biotech Co., Ltd., Shanghai, China. E. coli ATCC 25922 strains was used as negative controls.