Effects of long-term exposure to an antibiotic in zebrafish: from the organism to the microbiome level

Due to high food demand, aquaculture is one of the fastest growing industries. Hence, to supply all the needs, organisms are commonly grown under stress conditions (e.g. overcrowded) which favor the emergence and spread of infectious diseases. To combat this problem, broad-spectrum antibiotics such as oxytetracycline (OTC) are used. Since organisms are not able to absorb all the antibiotic consumed, it is known that a large part of these compounds is excreted in its active form and released to the environment. Once in the environment, antibiotics can have effects on non-target organisms such as fish, their microbiome, and the bacterial communities in the water. Thus, this work aimed to study, the impact of antibiotics, namely OTC, on zebrafish in an integrated analysis at various levels of biological organization. For this purpose, in a first experience, adult zebrafish were exposed to OTC for two months via water. The analysis of the effects was performed at a sub-individual level (e.g. enzyme activity), individual (e.g. behavior) and microbiome (e.g. structure, function, and selection of bacteria resistant to OTC and resistance genes). Then, to verify the reversibility of effects in a non-continuous exposure scenario, after a long-term exposure period, organisms were transferred and kept in clean water for one month. In this experiment, the analysis of the effects was performed at a sub-individual level (e.g. enzyme activity), and in the microbiome (e.g. structure, function, and selection of bacteria resistant to OTC and resistance genes). Analyses during the exposure (five days and two months of exposure) and in the post-exposure period (five days and one month of post-exposure) were also performed. During the exposure period, our results revealed that OTC had an impact in organisms at several biological organization levels. At a sub-individual level, a decrease in the detoxification mechanisms and cellular energy available was observed. At individual level, an increase of stress response was observed through the increase of hyperactivity of fish. At bacterial communities’ level, OTC exposure induced changes at structure and functional level in both fish gut and water. For instance, in water samples ou results suggested the selection of some bacterial phylotypes that play a role in the nitrogen cycle. Regarding the post-exposure period, in both parameters analyzed (energetic reserves and bacterial communities’ structure and function) differences between the exposed and control group were minimized indicating a recovery in the fish gut and water. The role of OTC in the selection of antibiotic-resistant bacteria and antibiotic resistance genes was also verified. Namely, the selection of resistant bacteria indicated as potential pathogens to humans and fish was observed. Moreover, the pathogenic potential of some isolates was tested and confirmed in vivo. Overall, our work demonstrated that the water contamination with the antibiotic has an impact in zebrafish with consequences at several levels analyzed. The use of sensitive endpoints, that may act like early warning signals, allowed to assess effects at low concentrations. The effects observed at organisms’ level (e.g. behavior change, energy reserves) can compromise fish ability of prey capture and predator avoidance, leading to further consequences at population level. Moreover, the selection of potential pathogens and/or bacterial phylotypes related with nitrogen cycle may have an impact at ecosystem level. Hence our work reinforces the importance of interdisciplinary studies for a deeper understanding of the real impact of antibiotics in the environment.