摘要

消毒废水流出物含有复杂的生物分子混合物（包括DNA）。如果传递抗生素抗性的完整基因在消毒过程中存活，环境细菌可能会将其吸收。我们用430mJ / cm2剂量的UV254处理含有氨苄青霉素抗性基因blaTEM-1和四环素抗性基因tetA的质粒pWH1266，研究了baylyi不动杆菌获得这些基因的能力。质粒每log10转化效率损失约20-25mJ / cm2。我们使用凝胶电泳和qPCR对质粒DNA降解进行监测，包括短扩增子（~200bps，代表常用于环境监测的ARG扩增子长度）和长扩增子（800-1200bps，旨在覆盖整个抗性基因）。qPCR测量的结果看，UV254处理导致的转化损失的速率分别约是用短扩增子基因降解速率20倍，约是长扩增子基因降解速率2倍。当外推以计算整个pWH1266质粒的长度时，qPCR速率常数比用转化测定法测量的速率常数大2-7倍。凝胶电泳结果证实DNA裂解不是主要的失活机制。总之，我们的研究结果表明，qPCR保守地测量了UV254处理后环境细菌转化的基因的潜力。

Disinfected wastewater effluent contains a complex mixture of biomolecules including DNA. If intact genes conveying antibiotic resistance survive the disinfection process, environmental bacteria may take them up. We treated plasmid pWH1266, which contains ampicillin resistance gene blaTEM-1 and tetracycline resistance gene tetA, with UV254 doses up to 430 mJ/cm2 and studied the ability of those genes to be acquired by Acinetobacter baylyi. The plasmids required approximately 20–25 mJ/cm2 per log10 loss of transformation efficiency. We monitored plasmid DNA degradation using gel electrophoresis and qPCR with both short amplicons (∼200 bps, representative of ARG amplicon lengths commonly used for environmental monitoring) and long amplicons (800–1200 bps, designed to cover the entire resistance genes). The rate of transformability loss due to UV254 treatment was approximately 20× and 2× larger than the rate of gene degradation measured with the short and long amplicons qPCR, respectively. When extrapolated to account for the length of the entire pWH1266 plasmid, the qPCR rate constants were 2–7× larger than the rate constants measured with transformation assays. Gel electrophoresis results confirmed that DNA cleavage was not a major inactivating mechanism. Overall, our results demonstrate that qPCR conservatively measures the potential for a gene to be transformed by environmental bacteria following UV254 treatment.

来源http://pubs.acs.org/doi/abs/10.1021/acs.est.7b01120