三地水源水中抗生素和抗性基因检出情况及相关性分析

Positive antibiotic and resistance genes in source water of three regions and correlation analysis

  • 摘要:
    背景 我国是抗生素生产和使用大国。抗生素的滥用可使水环境中的细菌获得耐药性,并且促进抗性基因(ARGs)的产生与传播。抗生素导致的耐药菌问题日益严重,已成为全球关注的公共安全问题。水环境作为抗生素和ARGs的巨大储存库,研究水体中抗生素污染水平和ARGs的现状对于保护水源和优化饮用水的生物安全至关重要。
    目的 评价典型水源水中抗生素和ARGs的检出情况,并探讨抗生素和ARGs之间的关系。
    方法 于2020年丰水期(8—10月间)在黑龙江、辽宁、湖北三省采集水源水样。三地各采集10份水样,共30份样本。采用超高效液相色谱串联质谱法检测大环内酯类、喹诺酮类、磺胺类、四环素类、β-内酰胺类5类抗生素;采用实时荧光定量PCR(qPCR)对整合子基因(Intl1)、16S rRNA及6种ARGs进行了检测。ARGs包括1种大环内酯类(ermB)、1种β内酰胺类(blaTEM)、2种四环素类(tetCtetQ)和2种磺胺类(sul1、sul2)。
    结果 三地水源水中抗生素的检出种类不同,并且相同抗生素在不同水源水中的质量浓度(后简称为浓度)范围也不同(P<0.05)。5大类抗生素中,A地的检出浓度范围为0.11~418.80 ng·L−1,B地为0.12~23.23 ng·L−1,C地为4.69~285.75 ng·L−1。三地水源水中6种ARGs的检出率均达到了100%。A地ARGs的绝对丰度范围为22.56~94355.91(每毫升拷贝数,后同),B地绝对丰度的范围为27.99~80584.32,C地绝对丰度的范围为41.99~111068.19。在三地水源水不同ARGs中,blaTEM的绝对丰度较高,次之是sul1sul2。此外,三地水源水中Intl1的绝对丰度也处于较高水平。相关性分析显示,三地水源水中各ARGs绝对丰度之间基本呈正相关;各类抗生素与其对应ARGs之间对应关系不明显;整合子Intl1sul1sul2P< 0.05)之间均呈正相关关系。
    结论 各地水源水中抗生素种类与浓度、ARGs丰度等检出情况差异较大;抗生素和ARGs的相关关系不确定;整合子Intl1可能在磺胺类抗性基因的水平转移过程中起重要作用。

     

    Abstract:
    Background China is a big country in the production and use of antibiotics. The abuse of antibiotics enables bacteria in water environment to acquire resistance, and promotes the generation and spread of antibiotics resistance genes (ARGs). The problem of antibiotic-resistant bacteria is increasingly serious and has become a public security issue of global concern. Water environment is a huge reservoir of antibiotics and ARGs. It is of great significance to study the pollution of antibiotics and ARGs in water to protect water sources and optimize the biosecurity of drinking water.
    Objective To evaluate the detection of antibiotics and ARGs in typical water sources, and to explore the relationship between antibiotics and ARGs.
    Methods Water samples were collected in Heilongjiang, Liaoning, and Hubei provinces during the wet season (from August to October) in 2020. Ten water samples were collected from each of the three places, and a total of 30 water samples were collected in this study. Five kinds of antibiotics, including macrolides, quinolones, sulfonamides, tetracycline, and β-lactam, were detected by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The integron (Intl1), 16S rRNA, and 6 kinds of ARGs were detected by quantitative real-time PCR (qPCR). The ARGs include one macrolide ARGs (ermB), one β-lactam ARGs (blaTEM), two tetracycline ARGs (tetC, tetQ), and two sulfonamide ARGs (sul1, sul2).
    Results The types of detected antibiotics varied by the three regions, and the concentration ranges of the same antibiotics varied by the three regions (P<0.05). The concentration ranges of selected five kinds of antibiotics were 0.11-418.80 ng·L−1 in region A, 0.12-23.23 ng·L−1 in region B, and 4.69-285.75 ng·L−1 in region C, respectively. The detection rates of all six ARGs were 100%. The absolute abundance of ARGs in region A ranged from 22.56 to 94355.91 copies·mL−1, that in region B ranged from 27.99 to 80584.32 copies·mL−1, and that in region C ranged from 41.99 to 111068.19 copies·mL−1. The absolute abundance of blaTEM was higher among the ARGs, followed by sul1 and sul2. In addition, the absolute abundance of Intl1 was also at a high level. The results of correlation analysis showed that the abundance of ARGs was positively correlated with each other. There was no correlation between specific antibiotics and corresponding ARGs. There was a positive correlation between Intl1 and sul1 or sul2 (P<0.05).
    Conclusion The types and concentrations of antibiotics and the abundance of ARGs in source water vary greatly in the study areas. The association between antibiotics and ARGs is uncertain. Intl1 may play an important role in the horizontal transfer of sulfonamide resistance genes.

     

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