新抗生素耐药菌的出现对人类健康构成了严重威胁，这主要归因于抗生素耐药基因( ARGs )的进化和传播。该文提出了一种宏基因组学指导的策略，包括宏基因组分析和功能验证，用于从ARG传播热点如污水处理厂( WWTPs )和动物粪便中快速鉴定新的ARG。我们使用抗生素抗性基因数据库注释来自污泥和鸡粪便的宏基因组的76个推定的β-内酰胺抗性基因。在76个候选基因中，与已知β-内酰胺酶具有40～70 %氨基酸同一性的25个靶基因进行PCR克隆。进一步证实了它们对4种β-内酰胺类抗生素的耐药性。此外，已验证的ARG被作为参考序列，对8个环境样品中的新ARG进行了鉴定，表明需要利用已验证的新ARG序列对环境样品中的ARG谱重新进行检测。这种宏基因组学导向的途径在初始筛选过程中不依赖于ARGs的活性，并且可以特异性地选择新的ARG序列用于功能验证，这使得其适用于来自环境宏基因组的新型ARG的高通量筛选。

      Emergence of new antibiotic resistance bacteria poses a serious threat to human health, which is largely attributed to the evolution and spread of antibiotic resistance genes (ARGs). In this work, a metagenomics-guided strategy consisting of metagenomic analysis and function validation was proposed for rapidly identifying novel ARGs from hot spots of ARG dissemination, such as wastewater treatment plants (WWTPs) and animal feces. We used an antibiotic resistance gene database to annotate 76 putative β-lactam resistance genes from the metagenomes of sludge and chicken feces. Among these 76 candidate genes, 25 target genes that shared 40~70% amino acid identity to known β-lactamases were cloned by PCR from the metagenomes. Their resistances to four β-lactam antibiotics were further demonstrated. Furthermore, the validated ARGs were used as the reference sequences to identify novel ARGs in eight environmental samples, suggesting the necessity of re-examining the profiles of ARGs in environmental samples using the validated novel ARG sequences. This metagenomics-guided pipeline does not rely on the activity of ARGs during the initial screening process and may specifically select novel ARG sequences for function validation, which make it suitable for the high-throughput screening of novel ARGs from environmental metagenomes.

https://link.springer.com/article/10.1007/s00253-017-8343-0