Dose-dependent impact of oxytetracycline on the veal calf microbiome and resistome

Background: Antibiotic therapy is commonly used in animal agriculture. Antibiotics excreted by the animals can contaminate farming environments, resulting in long term exposure of animals to sub-inhibitory levels of antibiotics. Little is known on the effect of this exposure on antibiotic resistance. In this study, we aimed to investigate the long term effects of sub-inhibitory levels of antibiotics on the gut microbiota composition and resistome of veal calves in vivo.Forty-two veal calves were randomly assigned to three groups. The first group (OTC-high) received therapeutic oral dosages of 1 gram oxytetracycline (OTC), twice per day, during five days. The second group (OTC-low) received an oral dose of OTC of 100 – 200 µg per day during seven weeks, mimicking animal exposure to environmental contamination. The third group (CTR) did not receive OTC, serving as unexposed control. Antibiotic residue levels were determined over time. The temporal effects on the gut microbiota and antibiotic resistance gene abundance was analysed by metagenomic sequencing.Results: In the therapeutic group, OTC levels exceeded MIC values. The low group remained at sub-inhibitory levels. The control group did not reach any significant OTC levels. 16S rRNA gene-based analysis revealed significant changes in the calf gut microbiota. Time-related changes accounted for most of the variation in the sequence data. Therapeutic application of OTC had transient effect, significantly impacting gut microbiota composition between day 0 and day 2. By metagenomic sequence analysis we identified six antibiotic resistance genes representing three gene classes (tetM, floR and mel) that differed in relative abundance between any of the intervention groups and the control. qPCR was used to validate observations made by metagenomic sequencing, revealing a peak of tetM abundance at day 28-35 in the OTC-high group. No increase in resistance genes abundance was seen in the OTC-low group.Conclusions: Under the conditions tested, sub-therapeutic administration of OTC did not result in increased tetM resistance levels as observed in the therapeutic group.研究背景：抗生素治疗在畜牧养殖中应用广泛。动物体内未代谢完全的抗生素会随排泄物污染养殖环境，致使动物长期暴露于亚抑菌浓度的抗生素环境中。目前人们对这类暴露对抗生素耐药性的影响尚不清楚。本研究旨在体内探究亚抑菌浓度抗生素长期暴露对肉用犊牛肠道菌群组成及耐药组（resistome）的影响。本研究将42头肉用犊牛随机分为三组。第一组（高剂量土霉素组，OTC-high）每日口服给药1g土霉素（oxytetracycline, OTC），每日2次，连续给药5天，为治疗给药组。第二组（低剂量土霉素组，OTC-low）每日口服给予100~200 µg OTC，持续7周，模拟动物暴露于环境抗生素污染的状态。第三组为空白对照组（CTR），不给予任何OTC。研究期间定期检测抗生素残留水平，并通过宏基因组测序分析肠道菌群及抗生素耐药基因丰度的时序变化特征。研究结果：治疗给药组的土霉素浓度超过了最低抑菌浓度（minimum inhibitory concentration, MIC），低剂量暴露组始终维持在亚抑菌浓度水平，空白对照组未检测到显著的土霉素残留。基于16S rRNA基因的菌群分析显示，犊牛肠道菌群结构发生了显著变化，时序相关的差异解释了测序数据中的大部分变异。土霉素治疗给药仅产生短暂影响，在第0天至第2天期间显著改变了肠道菌群组成。通过宏基因组测序分析，本研究共鉴定出6个抗生素耐药基因，分属3个基因家族（tetM、floR及mel），上述耐药基因的相对丰度在各干预组与空白对照组间均存在显著差异。采用实时荧光定量PCR（qPCR）对宏基因组测序结果进行验证，结果显示高剂量土霉素组的tetM基因丰度在第28~35天达到峰值，而低剂量土霉素组未出现耐药基因丰度的显著升高。研究结论：在本实验测试条件下，亚治疗剂量的土霉素给药并未像治疗给药组那样出现tetM耐药基因丰度的升高。