摘要

水生态系统日益受到人类活动的威胁，例如废水排放和农场经营。采用qPCR和扩增子下一代测序等方法评价人类活动对环境生物风险的影响。然而，这些方法不能提供目标物种的基因组信息，这对从基因组角度进行风险评估至关重要。在这里，我们开发了一种整合宏基因组分析和流式细胞术的新方法，以鉴定和量化环境中潜在的致病性抗生素抗性细菌（PARB；同时携带抗生素抗性基因（ARGS）和毒力因子基因（VFGS））。这些抗生素抗性细菌由于它们的感染能力和抗药性而引起特别关注。基于PARB的丰度/密度，我们评估了受城市排水和农业径流影响的河流中的微生物风险。我们在上游（山区）采集了样本作为对照。结果表明，使用我们的方法对所调查的占优势的的PARB（33）与已知的致病类群有81.8%的相关性。此外，占优势的假单胞菌基因组中的ARGs与 VFGS基因组内共存的模式（71个PARB中的20个）与来自NCBI RefSeq数据库最密切相关的假单胞菌基因组具有高度的相似性。这些结果反映了这种环境样品中（潜在）病原体鉴定方法可接受的可靠性。根据PARB密度，来自农业区的样品的微生物风险显著高于来自城市地区的样品。我们推测，这是由于农业中抗生素使用率较高，以及在抗生素选择压力下基因组内ARGs-VFGs协同进化所致。该研究为水生环境中PARB的识别和定量提供了一种替代方法，这种方法可用于评估微生物风险。

Aquatic ecosystems have been increasingly threatened by anthropogenic activities, e.g., wastewater discharge and farm operation. Several methods are adopted to evaluate the effects of anthropogenic activities on biological risk in the environment, such as qPCR and amplicon next-generation sequencing. However, these methods fall short of providing genomic information of target species, which is vital for risk assessment from genomic aspect. Here, we developed a novel approach integrating metagenomic analysis and flow cytometry to identify and quantify potential pathogenic antibiotic resistant bacteria (PARB; carrying both antibiotic resistance genes (ARGs) and virulence factor genes (VFGs)) in the environment, which are of particular concern due to their infection ability and antibiotic resistance. Based on the abundance/density of PARB, we evaluated microbiological risk in a river impacted by both municipal drainage and agriculture runoff. We collected samples upstream (mountainous area) as the control. Results showed that 81.8% of dominant PARB (33) recovered using our approach were related to known pathogenic taxa. In addition, intragenomic ARGs-VFGs coexistence patterns in the dominant Pseudomonas genomes (20 out of 71 PARB) showed high similarity with the most closely related Pseudomonas genomes from the NCBI RefSeq database. These results reflect acceptable reliability of the approach for (potential) pathogen identification in environmental samples. According to the PARB density, microbiological risk in samples from the agricultural area was significantly higher than in samples from the urban area. We speculated that this was due to the higher antibiotic usage in agriculture as well as intragenomic ARGs-VFGs co-evolution under antibiotic selective pressure. This study provides an alternative approach for the identification and quantification of PARB in aquatic environments, which can be applied for microbiological risk assessment.

https://www.sciencedirect.com/science/article/pii/S0043135419309340