Impact of the antibiotic doxycycline on the D. magna reproduction, associated microbiome and antibiotic resistance genes in treated wastewater conditions.

Wastewater Treatment Plant (WWTP) effluents are important sources of antibiotics, antibiotic resistance genes (ARGs) and resistant bacteria that threaten aquatic biota and human heath. Antibiotic effects on host-associated microbiomes, spread of ARGs and the consequences for host health are still poorly described. This study investigated changes of the Daphnia magna associated microbiome exposed to the recalcitrant antibiotic doxycycline under artificial reconstituted lab water media (lab water) and treated wastewater media. D. magna individual juveniles were exposed for 10 days to treated wastewater with and without doxycycline, and similarly in lab water. We analysed 16 S rRNA gene sequences to assess changes in community structure, monitored Daphnia offspring production and quantified ARGs abundances by qPCR from both Daphnia and water (before and after the exposure). Results showed that doxycycline and media (lab water or wastewater) had a significant effect modulating Daphnia-associated microbiome composition and one of the most discriminant taxa was Enterococcus spp. Moreover, in lab water, doxycycline reduced the presence of Limnohabitans sp., which are dominant bacteria of the D. magna-associated microbiome and impaired Daphnia reproduction. Contrarily, treated wastewater increased diversity and richness of Daphnia-associated microbiome and promoted fecundity. In addition, the detected ARG genes in both lab water and treated wastewater medium included the qnrS1, sul1, and blaTEM, and the integron-related intI1 gene. The treated wastewater contained about 10 times more ARGs than lab water alone. Furthermore, there was an increase of sul1 in Daphnia cultured in treated wastewater compared to lab water. In addition, there were signs of a higher biodegradation of doxycycline by microbiomes of treated wastewater in comparison to lab water. Thus, results suggest that Daphnia-associated microbiomes are influenced by their environment, and that bacterial communities present in treated wastewater are better suited to cope with the effects of antibiotics.

Impact of organic soil amendments in antibiotic levels, antibiotic resistance gene loads, and microbiome composition in corn fields and crops.

The potential spreading of antibiotic resistance genes (ARG) into agricultural fields and crops represent a fundamental limitation on the use of organic fertilization in food production systems. We present here a study of the effect of spreading four types of organic soil amendments (raw pig slurry, liquid and solid fractions, and a digested derivative) on demonstrative plots in two consecutive productive cycles of corn harvest (Zea mays), using a mineral fertilizer as a control, following the application of organic amendments at 32-62 T per ha (150 kg total N/ha) and allowing 5-8 months between fertilization and harvest. A combination of qPCR and high-throughput 16S rDNA sequencing methods showed a small, but significant impact of the fertilizers in both ARG loads and microbiomes in soil samples, particularly after the second harvesting cycle. The slurry solid fraction showed the largest impact on both ARG loads and microbiome variation, whereas its digestion derivatives showed a much smaller impact. Soil samples with the highest ARG loads also presented increased levels of tetracyclines, indicating a potential dual hazard by ARG and antibiotic residues linked to some organic amendments. Unlike soils, no accumulation of ARG or antibiotics was observed in corn leaves (used as fodder) or grains, and no grain sample reached detection limits for neither parameter. These results support the use of organic soil amendments in corn crops, while proposing the reduction of the loads of ARGs and antibiotics from the fertilizers to greatly reduce their potential risk.