Table_1_Effect of high-copper diet on transference of blaCTX−M genes among Escherichia coli strains in rats' intestine.DOCX

Both ceftiofur (CTO) and high copper are widely utilized in animal production in China, and the occurrence of CTX-M-carrying Escherichia coli in food-producing animals is increasing. There are some specific associations between in-feed high-level copper and antibiotic resistance, but research in Gram-negative bacteria such as E. coli remains scarce. This study aimed to evaluate the effect of high-copper diet on the horizontal transfer of blaCTX−M−1 among E. coli. A total of 32 male SPF rats aged 21 days were randomly assigned to the following four groups: control (6 mg/kg in-feed copper, C−), high copper (240 mg/kg in-feed copper, H−), CTO (6 mg/kg in-feed copper with oral CTO administration, C+), and high copper plus CTO (240 mg/kg in-feed copper with oral CTO administration, H+). All rats were orally inoculated with an E. coli strain harboring a conjugative plasmid carrying blaCTX−M−1, and the C+ and H+ groups were given 10 mg/kg of body weight (BW) CTO hydrochloride at 26, 27, and 28 days, while the C− and H− groups were given salad oil at the same dose. Fecal samples collected at different time points were used for the enumeration of E. coli on Mac plates or for molecular analysis using PCR, pulsed-field gel electrophoresis (PFGE), S1-PFGE, and Southern-blot hybridization. The results showed that the number of the blaCTX−M−1 gene in the H− group was higher and that the loss speed of this gene was slower compared with the C− group. After administration of CTO, the counts of cefotaxime-resistant E. coli were significantly higher in the C+ group than that in the corresponding control group (C+ vs. C−; H+ vs. H−). In the in vitro test, the results showed that the transfer rates of the conjugation induced by the H− (12 mmol/L) group were significantly higher than that of low copper (2 mmol/L) group. The indigenous sensitive isolates, which were homologous to the blaCTX−M-positive isolates of rat feces, were found by PFGE. The further analysis of S1-PFGE and Southern-blot hybridization confirmed that the blaCTX−M−1 gene in new transconjugants was derived from the inoculated strain. Taken together, high-copper diet facilitates the horizontal transfer and maintenance of the resistant genes in the intestine of rats, although the effects of antibiotics on bacterial resistance appearance and maintenance are more obvious.

头孢噻呋（ceftiofur, CTO）与高铜饲料在中国畜禽养殖中应用广泛，而食品动物中携带CTX-M型基因的大肠埃希菌（Escherichia coli, E. coli）检出率呈上升趋势。饲用高铜与抗生素耐药性之间存在特定关联，但针对大肠埃希菌等革兰氏阴性菌（Gram-negative bacteria）的相关研究仍较为匮乏。本研究旨在探讨高铜饲料对大肠埃希菌中blaCTX-M-1基因水平转移的影响。本研究共选用32只21日龄雄性无特定病原体（Specific Pathogen Free, SPF）大鼠，随机分为4组：对照组（饲用铜含量6 mg/kg，C−组）、高铜组（饲用铜含量240 mg/kg，H−组）、头孢噻呋组（饲用铜含量6 mg/kg+口服头孢噻呋，C+组）以及高铜+头孢噻呋组（饲用铜含量240 mg/kg+口服头孢噻呋，H+组）。所有大鼠均经口接种携带blaCTX-M-1基因的接合性质粒的大肠埃希菌菌株；C+组与H+组于第26、27、28天按10 mg/kg体重（body weight, BW）给予盐酸头孢噻呋，C−组与H−组则以相同剂量给予色拉油作为对照。于不同时间点采集粪便样本，分别用于麦康凯平板（MacConkey plates, Mac平板）计数大肠埃希菌，以及采用聚合酶链式反应（Polymerase Chain Reaction, PCR）、脉冲场凝胶电泳（Pulsed-field Gel Electrophoresis, PFGE）、S1-PFGE及Southern印迹杂交（Southern blot hybridization）进行分子生物学分析。结果显示，与C−组相比，H−组粪便中blaCTX-M-1基因的检出量更高，且该基因的丢失速度更慢。给予头孢噻呋后，C+组的头孢噻肟耐药大肠埃希菌计数显著高于其对应对照组（C+组 vs. C−组；H+组 vs. H−组）。体外试验结果显示，12 mmol/L高铜组诱导的接合转移率显著高于2 mmol/L低铜组。通过PFGE分析发现，大鼠粪便中存在与blaCTX-M阳性分离株同源的本土敏感分离株。进一步通过S1-PFGE及Southern印迹杂交分析证实，新型接合子中的blaCTX-M-1基因均来源于接种的菌株。综上，尽管抗生素对细菌耐药性的出现与维持作用更为显著，但高铜饲料可促进大鼠肠道内耐药基因的水平转移与维持。