January 30, 2024

In January, the AquaticPollutants research project ARENA performed sampling activities in a land-based recirculating aquaculture commercial fish farm located close to the Orbetello lagoon (South Tuscany, Italian West Coast). The Recirculating Aquaculture Systems (RAS) facility is composed of 45 cages containing gilt-head sea bream (Sparus aurata), European seabass (Dicentrarchus labrax) and goldmouth croaker (Argyrosomus regius,). The latter was not in the project's focus.

Our colleagues from ARENA selected ten sites in which they collected water, sediment, feeding and fishes. From each fish they isolated skin, gill, gut (anterior, mid and hind gut were separated; gut tissues were obtained by aseptic dissection and the intestinal content was collected by gentle squeezing) and fillet.

The samples were obtained by dissecting fish specimens using sterile scissors, immediately placed in sterile tubes, and stored at − 20 °C until further analyses. Surface water samples were collected using sterile plastic bottles, filtered and stored at − 20 °C until processing; the entire membrane filters were used for DNA extraction. Surface sediment samples were collected by a grab sampler, stored in sterile tubes at -20°C until the DNA extraction. Finally, 10 g (in duplicate) of feeding samples has been collected into sterile tube, stored at − 20 °C until the DNA extraction.

DNA was extracted from all the samples collected using the DNeasy PowerSoil Kit (Qiagen) and
quantified. Currently, an aliquot from each extracted DNA sample from environmental samples
has been sent to an external sequencing service for shotgun metagenomic sequencing; a second
aliquot of DNA has been used for qPCR analyses on selected antibiotic resistance genes to unveil the information on the presence and levels of antibiotic resistance in microbial communities sampled in open-sea and RAS aquaculture settings.

The molecular results will be interpreted considering additional analyses. Here, the focus lies on the concentrations of antibiotic residues and microbial activities in the considered areas and samples. In particular, the analysis of antibiotic resistance contamination is being performed in:

(1) environmental samples, such as seawater and sediments and in the provided feedings, to
understand in which environmental compartment the selective pressure exerted by ABs is more
effective;
(2) in the farmed fish, to clarify whether antibiotic resistance is transferred to the food and to
hypothesize the risk posed for human food consumption.