We characterized for the first time the effects of bacterial infection with Photobacterium damselae ssp. piscicida and Vibrio harveyi and treatment with flumequine in the skin microbiota of seabass fingerlings. Most of our predictions were confirmed with one important exception; contrary to our expectations and most of the previous literature, both core microbiota and microbial diversity increased with the onset of disease. However, dysbiosis was accompanied by an increase in the abundance of potential pathogenic and opportunistic taxa.
Disease effects in skin microbiota of seabass fingerlings: healthy vs diseased states
Impacts on microbial diversity, richness and evenness caused by infection by bacterial pathogens and parasites have been described in some fish [in the skin of rainbow trout infected with Ichthyophthirius multifilis, 14; gut of Asian seabass infected with Tenacibaculum singaporense, 16; skin of Atlantic salmon infected with Lepeophtheirus salmonis, 6; gut of grass carp with enteric infection, 12; gut of brown trout infected with Tetracapsuloides bryosalmonae, 15; skin of orbicular batfish infected with Tenacibaculum maritimum, 13; gut and stomach of rainbow trout infected with Caligus lacustri, 11; and skin of adult seabass infected with Photobacterium damselae, 7]. Dysbiosis was reported on the vast majority of these studies through decreases in fish microbial diversity and increases in pathobionts. Even though an increase in diversity was observed in the present study, the direction of the changes in the abundance of key microbial taxa indicates dysbiosis occurred in the skin microbiome of seabass fingerlings. In the present study, Vibrio (that encompasses one of the etiological agents of infection), and two other unidentified genera belonging to families with opportunistic taxa, Flavobacteriaceae and Vibrionaceae , increased their abundance in the diseased state. Another genus that increased in abundance in diseased fish was Aureispira, previously found to be highly abundant in the intestinal microbiota of grouper juveniles after iridovirus infections . Similar increases in bacterial diversity after disease have been already described in other fish [e.g., 6, 12, 14, 15], indicating that changes in microbial diversity cannot be readily expected and growth or decline of specific taxa easily predicted.
Alterations to the core microbiota in response to infection were also previously reported in the skin of adult European seabass infected with Photobacterium damselae , and in the yellowtail kingfish infected with with enteritis . In the present study, a Photobacterium damselae ASV was 100% prevalent in the diseased state. However, its mean abundance remained unaltered between the healthy and disease states, suggesting the skin was only indirectly affected by this pathogen. These results are in line with our previous study showing that infection caused by P. damselae can lead to dysbiosis of skin microbiota of farmed seabass despite no increase in abundance . Similar results were also obtained by Legrand et al. , who described skin and gill dysbiosis in the yellowtail kingfish during enteritis, a gut disease.
Microbial structure of the skin microbiota was also significantly affected by disease. Although samples of diseased fish were collected on the day prior to disease onset, only a few samples from the diseased state (7 out of 30) clustered within the healthy group, confirming that significant taxonomic changes had occurred in most individuals analyzed. These results suggest that some properties of the skin mucous that allowed certain phylogenetically related taxa to thrive in the skin may have been altered by disease, consequently affecting resident microbiota. However, despite the increase in microbial diversity and changes in structure driven by disease, microbial metabolic functions remained unaltered. This suggests that the increase in diversity observed between healthy and diseased states was due to colonization by bacteria with similar functions .
Flumequine effects in skin microbiota of seabass fingerlings: diseased vs treatment states
In the present study, microbial diversity was observed to increase on the 8th day of treatment with flumequine. However, as expected, administration of flumequine resulted in a decrease in abundance of both etiological agents of disease in this study. This is unsurprising given the reported sensitivity of both species to this antibiotic . Importantly, this treatment led to an increase of potentially harmful Flavobacteriaceae . Interestingly, the genus Alteromonas, which has been shown to exhibit antibacterial activity against fish pathogens, including Photobacterium damselae and several Vibrio spp. , and resistance against amoxicillin, erythromycin and gentamicin , increased in abundance during antibiotic treatment. Microbial disruptions have been reported after oxytetracycline and rifampicin treatment in microbiota of adult fish [e.g., 7, 22–24], and after streptomycin, ciprofloxacin or oxytetracycline treatment on earlier life fish stages [e.g., zebrafish larvae, 25, 44]. Although an increase in fish microbial diversity caused by antibiotic administration is less common, it has been reported before [e.g., 7, 44]. Indeed, in the studies where diversity decreased after antibiotic exposure, there were no pre-existing health conditions. On the other hand, in this study as well as in Rosado et al. , disease had occurred, and in the study by López Nadal et al. , fish were immersed with the anti-nutritional compound saponin before antibiotic treatment. This suggests pre-existing disease/microbiota disruption and antibiotics may have a compound effect on microbial diversity.
Significant changes in the potential function of the skin microbiota were detected after antibiotic treatment. Specifically, the degradation of carbohydrates and secondary metabolites were significantly enriched during antibiotic treatment. However, the production of carbohydrates and secondary metabolites is linked to the protective role of the microbiota [e.g., 1, 45]. For example, carbohydrates are directly related to specific cell-cell adhesion, modulating microbial binding to the mucus . It is suggested that carbohydrate synthesis by the human microbiota helps establish symbiosis with microbial commensals and aids pathogenic evasion . Furthermore, production of secondary metabolites is one of the mechanisms by which commensal microbiota fight against pathogens . These results suggest a microbial response to antibiotics, which may ultimately have a negative effect on fish immunity.
Recovery of the skin microbiota in seabass fingerlings: healthy vs recovery states
Previous studies reported that short-term recovery of the microbiota of fish after antibiotic treatment does not lead to the diversity levels observed in the healthy state (e.g., 1-week recovery, ). In the present study, with the exception of Pielou’s evenness, diversity significantly increased between healthy and recovery states. Importantly, the abundance of Vibrio increased in the recovery state, indicating microbial balance may not have been fully obtained.
Microbial structure was also significantly different between the recovery and healthy states. However, closely related microbial structuring was found in fish from treatment and recovery states. Almost half of the enriched metabolic pathways during the recovery state were related to the same categories of pathways enriched during the treatment state (carbohydrate and secondary metabolite degradation), with significant differences from the healthy state. To the best of our knowledge, only the study by Brumlow et al.  has effectively measured the effects of 3-day antibiotic treatments (Tetracycline and Rifampicin) in the biochemical profile of the skin of Gambusia affinis. In this study the authors also report changes in community composition relative to pre-treatment, after 8 day recovery. However, unlike the present results, where significant changes to microbial function were predicted, the results of Brumlow et al.  indicated that the biochemical functions of microbiota were mostly reestablished after the 8 day recovery. Flumequine is a highly persistent antibiotic and it can take several weeks to be fully depleted from the blood and tissues of fish [48, 49]. This antibiotic has a slower depletion rate in the skin than in muscle or liver, and can be present in the skin 20 days after oral administration . Although a longer time frame would be necessary to evaluate whether full functional recovery of the skin microbiota does occur, our results highlight the high susceptibility of skin microbiota to antibiotic exposure.