Data_Sheet_1_Development of an in vitro biofilm model for the study of the impact of fluoroquinolones on sewer biofilm microbiota.xlsx

Sewer biofilms are likely to constitute hotspots for selecting and accumulating antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). This study aimed to optimize culture conditions to obtain in vitro biofilms, mimicking the biofilm collected in sewers, to study the impact of fluoroquinolones (FQs) on sewer biofilm microbiota. Biofilms were grown on coupons in CDC Biofilm Reactors®, continuously fed with nutrients and inoculum (1/100 diluted wastewater). Different culture conditions were tested: (i) initial inoculum: diluted wastewater with or without sewer biofilm, (ii) coupon material: concrete vs. polycarbonate, and (iii) time of culture: 7 versus 14 days. This study found that the biomass was highest when in vitro biofilms were formed on concrete coupons. The biofilm taxonomic diversity was not affected by adding sewer biofilm to the initial inoculum nor by the coupon material. Pseudomonadales, Burkholderiales and Enterobacterales dominated in the sewer biofilm composition, whereas in vitro biofilms were mainly composed of Enterobacterales. The relative abundance of qnrA, B, D and S genes was higher in in vitro biofilms than sewer biofilm. The resistome of sewer biofilm showed the highest Shannon diversity index compared to wastewater and in vitro biofilms. A PCoA analysis showed differentiation of samples according to the nature of the sample, and a Procrustes analysis showed that the ARG changes observed were linked to changes in the microbial community. The following growing conditions were selected for in vitro biofilms: concrete coupons, initial inoculation with sewer biofilm, and a culture duration of 14 days. Then, biofilms were established under high and low concentrations of FQs to validate our in vitro biofilm model. Fluoroquinolone exposure had no significant impact on the abundance of qnr genes, but high concentration exposure increased the proportion of mutations in gyrA (codons S83L and D87N) and parC (codon S80I). In conclusion, this study allowed the determination of the culture conditions to develop an in vitro model of sewer biofilm; and was successfully used to investigate the impact of FQs on sewer microbiota. In the future, this setup could be used to clarify the role of sewer biofilms in disseminating resistance to FQs in the environment.

污水生物膜（sewer biofilms）极有可能成为筛选并累积抗生素耐药菌（antibiotic-resistant bacteria, ARB）与抗生素耐药基因（antibiotic resistance genes, ARGs）的热点区域。本研究旨在优化培养条件，以获取模拟实际污水环境中采集的生物膜的体外生物膜（in vitro biofilms），从而探究氟喹诺酮类（fluoroquinolones, FQs）对污水生物膜微生物群落的影响。生物膜于CDC生物膜反应器（CDC Biofilm Reactors®）内的载片上培养，反应器持续通入营养物质与接种液（经1/100稀释的废水）。本研究测试了多种培养条件：(i) 初始接种液：含或不含污水生物膜的稀释废水；(ii) 载片材质：混凝土与聚碳酸酯；(iii) 培养时长：7天与14天。本研究发现，当体外生物膜在混凝土载片上形成时，生物量达到最高水平。初始接种液中添加污水生物膜，以及载片材质的选择，均不会对生物膜的分类多样性产生影响。假单胞菌目（Pseudomonadales）、伯克霍尔德菌目（Burkholderiales）与肠杆菌目（Enterobacterales）是污水生物膜群落的优势类群，而体外生物膜则主要由肠杆菌目构成。体外生物膜中qnrA、qnrB、qnrD与qnrS基因的相对丰度均高于污水生物膜。与废水及体外生物膜相比，污水生物膜的耐药组（resistome）香农多样性指数（Shannon diversity index）最高。主坐标分析（Principal Coordinates Analysis, PCoA）结果显示，样本可根据其类型进行区分；普氏分析（Procrustes analysis）结果表明，观测到的抗生素耐药基因丰度变化与微生物群落结构变化存在关联。最终选定的体外生物膜培养条件为：使用混凝土载片、以含污水生物膜的稀释废水作为初始接种液，以及14天的培养时长。随后，本研究在高、低两种氟喹诺酮类药物浓度下构建生物膜体系，以验证所建立的体外生物膜模型。氟喹诺酮类药物暴露对qnr基因的丰度无显著影响，但高浓度暴露会提升gyrA基因（密码子S83L与D87N）与parC基因（密码子S80I）的突变比例。综上，本研究明确了构建污水生物膜体外模型的最优培养条件，并成功利用该模型探究了氟喹诺酮类药物对污水生物膜微生物群落的影响。未来，该实验体系可用于进一步阐明污水生物膜在环境中传播氟喹诺酮类耐药性过程中所发挥的作用。