Introduction
Aquaculture is considered a hotspot for the emergence and spread of antimicrobial resistance (AMR) because t he coexistence of fish, bacteria and antibiotics in the aquatic environment provides the ideal conditions (Cabello et al, 2013). However, little is known on AMR in aquaculture compared to terrestrial farming. In particular, studies on the occurrence of AMR in recircula ting aquaculture systems (RAS) are very scarce, in spite of being the fish farming system of the future. The objectives of this study are 1) to describe the dynamics of selected indicator bacteria within a RAS (i.e. sources , persistence and diffusion ) and 2) to compare their AMR profiles to those from clinically important isolates in human medicine.
Materials and methods
This project is being performed at the RAS based at the vet school Oniris, Nantes , over six months (February-July 2022) . Rainbow trout is farmed year-long at this experimental station that uses domestic water and follows a tho rough water treatment: sand filtration, biological filtration and UV. The dynamics of AMR within this RAS are being
studied with a longitudinal study design, by tracking a selection of indicator bacteria in all compartments (water, sediment, fish , feed
and biofilm ). On a monthly basis, the following samples are collected : 7 water samples from different parts of the recirculating system (inclu ding each water treatment step, see Table 1 ), 1 sediment from water reservoir, 5 biofilms and 5 fecal samples from fish tanks . Each batch of fish feed distributed during the study period is sampled once. D ead fish are opportunistically sampled.
A ll the samples are tested for the presence of Escherichia coli , Pseudomonas and Aeromonas . E. coli is an indicator of fecal contamination from mammals and birds, whereas Aeromonas and Pseudomonas are ubiquitous aquatic bacteria. All three bacterial groups are good indicators of AMR in the environment as they are prone to accumulate and exchange AMR genes. Samples are plated on G lutamate S tarch Phenol Red agar (Merck, Germany) for detecting Aeromonas and Pseudomonas and on E osin Methylene Blue agar for detecting
E. coli
(Biokar, France),
then incubated at 37° C for 24-48 hours.
MALDI-TOF will be used
to confirm and identify isolates at species level. Confirmed isolates will be tested by the broth micro-dilution method to determine their Minimum Inibitory Concentration.
The antibiotics used in this antimicrobial susceptibility test
include Sulfamethoxazole, Trimethoprim, Ciprofloxacin, Tetracycline, Meropenem, Azithromycin, Nalidixic Acid, Cefotaxime, Chloramphenicol, Tigecycline, Ceftazidime, Colistin, Ampicillin, Gentamycin and Amikacine. For each of the bacterial groups studied (E. coli, Aeromonas, Pseudomonas ), 20 isolates from human clinical infections have been retrieved from the collection of the University Hospital (CHU Nantes) for comparison of AMR profiles.
Results and dis cussion
44 samples have been tested so far in two sampling events . Aeromonas is presumptively the most prevalent bacteria, being found in all compartments of the RAS and almost all the samples. Pseudomonas on the other hand seems less prevalent, especially in fish feces. No E. coli
was found, but coliforms were isolated from water, sediment, biofilm and feed. Further analyses are needed
to know whether coliform contamination from feed does persist in this RAS.
With regards to water, only domestic water is presumptively free of Aeromonas and Pseudomonas, meaning that water is the cleanest at the arrival to the station (Table 1). Fish and feed would be the main sources of these bacteria, which are likely to persist in this RAS. Interestingly, biofilm seems the most dynamic compartment of this system, presumptively shifting from being mainly Aeromonas -positive to being mainly Pseudomonas-positive, as water temperature rose from 14°C to 16°C.
References
Buschmann, A. H. (2013). Antimicrobial use in aquaculture re-examined: Its relevance to antimicrobial resistance and to animal and human health. Environmental Microbiology, 15:1917–1942. https://doi.org/10.1111/1462-2920.12134