Data_Sheet_1_The fate of sulfonamide resistance genes and anthropogenic pollution marker intI1 after discharge of wastewater into a pristine river stream.pdf

IntroductionCurrently there are sparse regulations regarding the discharge of antibiotics from wastewater treatment plants (WWTP) into river systems, making surface waters a latent reservoir for antibiotics and antibiotic resistance genes (ARGs). To better understand factors that influence the fate of ARGs in the environment and to foster surveillance of antibiotic resistance spreading in such habitats, several indicator genes have been proposed, including the integrase gene intI1 and the sulfonamide resistance genes sul1 and sul2.MethodsHere we used quantitative PCR and long-read nanopore sequencing to monitor the abundance of these indicator genes and ARGs present as class 1 integron gene cassettes in a river system from pristine source to WWTP-impacted water. ARG abundance was compared with the dynamics of the microbial communities determined via 16S rRNA gene amplicon sequencing, conventional water parameters and the concentration of sulfamethoxazole (SMX), sulfamethazine (SMZ) and sulfadiazine (SDZ).ResultsOur results show that WWTP effluent was the principal source of all three sulfonamides with highest concentrations for SMX (median 8.6 ng/l), and of the indicator genes sul1, sul2 and intI1 with median relative abundance to 16S rRNA gene of 0.55, 0.77 and 0.65%, respectively. Downstream from the WWTP, water quality improved constantly, including lower sulfonamide concentrations, decreasing abundances of sul1 and sul2 and lower numbers and diversity of ARGs in the class 1 integron. The riverine microbial community partially recovered after receiving WWTP effluent, which was consolidated by a microbiome recovery model. Surprisingly, the relative abundance of intI1 increased 3-fold over 13 km of the river stretch, suggesting an internal gene multiplication.DiscussionWe found no evidence that low amounts of sulfonamides in the aquatic environment stimulate the maintenance or even spread of corresponding ARGs. Nevertheless, class 1 integrons carrying various ARGs were still present 13 km downstream from the WWTP. Therefore, limiting the release of ARG-harboring microorganisms may be more crucial for restricting the environmental spread of antimicrobial resistance than attenuating ng/L concentrations of antibiotics.

当前，关于抗生素从污水处理厂（WWTP）排放至河流系统中的监管尚显不足，导致地表水成为抗生素及抗生素抗性基因（ARGs）的潜在储库。为深入探究影响环境中外源ARGs命运的因素，并促进此类生境中抗生素抗性传播的监测，已提出包括整合酶基因intI1以及磺胺类抗性基因sul1和sul2在内的多个指示基因。方法上，本研究采用定量PCR和长读长纳米孔测序技术，监测了这些指示基因以及作为第一类整合子基因盒存在的ARGs在河流系统中的丰度，该系统从原生水源至受WWTP影响的水体。通过16S rRNA基因扩增子测序确定的微生物群落动态、传统水质参数以及磺胺甲噁唑（SMX）、磺胺嘧啶（SMZ）和磺胺二甲基嘧啶（SDZ）的浓度，与ARGs的丰度进行了比较。结果显示，WWTP的排放物是三种磺胺类物质的主要来源，其中SMX的浓度最高（中位数8.6 ng/L），以及指示基因sul1、sul2和intI1，其相对于16S rRNA基因的中位相对丰度分别为0.55%、0.77%和0.65%。在WWTP下游，水质持续改善，包括磺胺类浓度降低、sul1和sul2的丰度减少以及第一类整合子中ARGs的数量和多样性降低。受WWTP排放物影响后，河流微生物群落部分恢复，并由微生物组恢复模型巩固。令人惊讶的是，在13公里长的河流段内，intI1的相对丰度增加了3倍，这表明基因内部发生了扩增。讨论中，本研究未发现低剂量的磺胺类物质在 aquatic environment 中能够刺激相应ARGs的维持甚至传播。尽管如此，携带各种ARGs的第一类整合子在WWTP下游13公里处依然存在。因此，限制携带ARGs的微生物的排放可能比降低抗生素ng/L浓度对于限制抗菌抗性在环境中的传播更为关键。