Transfer of Antibiotic Resistance Genes from a Waste Water Treated Plant Effluent to the Filter Feeding Zooplankton Daphnia Magna and its Associated Microbiome

Abstract

Waste Water Treatment Plant (WWTP) effluents are important sources of antibiotics, antibiotic resistant genes (ARGs) and other contaminants for the aquatic environment and biota. This study investigated the impact of the antibiotic doxycycline on the microbiome associated to the zooplanktonic filter feeder Daphnia magna, on its ability to reduce antibiotic resistant genes (ARGs), and the bacterial load from water and to incorporate them. D. magna individual juveniles were exposed for 10 days to a WWTP effluent with and without doxycycline and then transferred to clean water for an additional 4 days. Animals were fed with the microalgae Chlorella vulgaris, cultured in axenic conditions, plus an organic extract that promotes Daphnia reproduction. Results showed that in lab water, doxycycline impaired Daphnia reproduction and reduced the presence of Burkholderiales, which are dominant bacteria of the D. magna associated microbiome, whereas treated waste water promoted fecundity. The study of total and the diversity of 16s rDNA and of ARGs by means of qPCR and metabarcoding evidenced the presence of different bacteria and of ARGs in both lab water and waste water medium. The detected genes included the ARGs qnrS1, sul1, and blaTEM,, and the integron-related intI1 gene. The waste water treatment contained about 10 times more ARGs than lab water alone. The microbiome composition of Daphnia was well differentiated from that of water, as evidenced by studies of beta-diversity. There was an increase of sul1 in Daphnia cultured in waste water, along with an increase of the prevalence of the genera Ideonella, Flavobacterium and Sphingorabdus, likely candidates of carrying ARGs. Our results indicate that D. magna individuals by grazing on bacteria are able to incorporate ARGs from WWTP effluents and hence may act as a potential vector of transmission of antibiotic resistance genes.

Keywords: Daphnia, antibiotic resistance gene, wastewater, Bacteria, antibiotic