Introduction
Fish farming is striving to maximise sustainable production to meet the needs of a growing global human popula tion, however, disease is a major barrier to achieving this
. To address the problem of disease in a quaculture there has been, and continues to be, widespread use (and misuse) of antibiotics for both disease treatment and prevention
. Globally, antibiotic use is particularly prevalent in freshwater finfish culture
, however, it is unclear what influence antibiotic exposure may have on the fish microbiome under normal culture conditions , which is a critical component for maintaining health and resilience against disease. Here we assessed the effects of exposure to oxytetracycline (OTC) on the fish skin and pond water microbiomes; antibiotic residue fate; and development of antimicrobial resistance. This was performed as a mesocosm study in Khulna, Bangladesh to provide realistic environmental and microbial conditions that directly translate to aquaculture practices.
Materials and methods
Tilapia and carp (Oreochromis niloticus and Labeo rohita) were polycultured in earthen aquaculture ponds (3x control and 3x treatment ponds) and exposed to a typical medical treatment dose (100 mg/kg ) of OTC via the diet for five days . Sampling of water and fish skin swabs were collected prior to treatment , and subsequently at 2 , 9 and 23 days following the 5 day antibiotic exposure. To characterise the prokaryotic and microeukaryotic communities, amplicon sequencing libraries of pond water filters and fish skin swabs were prepared as previously described
. PCR-free metagenomes of pond water were also prepared to characterise the diversity of antimicrobial resistance genes selected in the resistome following antibiotic exposure. Sequencing of 16S V4 amplicons, metagenomes and 18S V9 amplicons was performed on the Illumina Novaseq SP- 250, S1-150 and Illumina MiSeq V2-150 respectively. To assess antibiotic residue fate, pond water was concentrated by Solid Phase Extraction and quantified by the multi- residue UPLC-MS/MS validated methodology of Holton and Kasprzyk-Hordern
. Generalised l inear mixed effects modelling was performed to statistically assess OTC residue fate and fish growth.
Results and discussion
T ilapia grew over the experimental period by 0.61 ± 0.31 g (95%-CI) p < 0.001 per day, with no observed difference between control and treatment groups p = 0.756, illustrating no toxic or beneficial effects of the antibiotic treatment on growth (Fig. 1A) . Throughout the experimental period, concentrations of OTC in water of control ponds remained low at 0.045 ± 0.018 µg/L (95%-CI) (Fig 1B) . Two days after the completion of exposure, OTC concentration reached 9.6 ± 2.11 µg/L (95%-CI) in dosed pond water, and the overall concentration of OTC in treatment ponds was significantly elevated compared to control ponds p < 0.001. At the final sampling point, 23 days post-exposure, the concentration of oxytetracycline in treatment ponds had returned to pre-exposure levels.
Ultra-deep sequencing of 16S V4 amplicons for 384 samples of fish skin and pond water yielded a median sequencing depth of 168,440 paired- end reads per sample. Additionally, 104 samples of pond water were sequenced as 18S V9 amplicons, resulting in a median sequencing depth of 56,184 paired-end reads. We will report the impact of antibiotic exposure on fish skin microbiome community structure and will correlate specific taxonomic shifts to measured oxytetracycline concentrations. In parallel, to assess the development of antimicrobial resistance following therapeutic use of antibiotics in aquaculture we performed shotgun metagenomic sequencing on 12 pond water metagenomes of control and treatment ponds. Sequencing generated a total of 750 million paired-end reads. Ongoing analysis is exploring the diversity and abundance of antimicrobial resistance genes, in addition to mobile genetic elements selected in the resistome, that will be reported upon at the meeting.
By characterizing animal and environmental health, as well as the development and spread of antimicrobial disease resistance, these analyses aim to provide a comprehensive understanding of the implications for antibiotic (mis)use in aquaculture within the one health framework.
References