**3.3 Effect of antibiotics**

In agricultural systems, the use of chemical products, especially antibiotics, has become widespread as a form of disease prevention and treatment [96]. While, in cattle breeding, antibiotics are preferably used as growth promoters [97], in aquaculture these properties have not been convincingly demonstrated. Historically, due to its intensive practice, the Chilean salmon industry is identified as one that uses more antibiotics per ton of harvested product [7]. In 2016, the amount of antibiotic used in the Chilean salmon industry reached 382.5 tons. The administration of antibiotics in aquaculture farms is mainly done through food, the remaining of which accumulates in the environment together with excretions [98]. Such accumulation of antibiotics in the marine sediments can persist for months, acting as a selection pressure that favors the establishment of resistant microorganisms that alter the endemic microbiota and the natural biogeochemical processes [99]. In Chile, this phenomenon is of great importance, since most of the salmon production is concentrated in the south of Patagonia, an area of high biological diversity.

The global trend in salmon production has been to reduce the use of chemical products to comply with biosecurity and animal welfare policies. In Chile, however, the use of antibiotics has continued to increase, accumulating an average of 343.4 tons of antibiotics per year (period 2005–2016) [100], 95% of which is used in marine fish farms. During this period, a high rate of infection by the intracellular bacterium *Piscirickettsia salmonis*, which causes the salmonid rickettsial syndrome (SRS), was observed, for which there is no effective vaccine or antibiotic treatment [101].

The main antibiotics used in Chile correspond to florfenicol and oxytetracycline. According to the national service of fishing and aquaculture from Chile (SERNAPESCA) in 2016, florfenicol and oxytetracycline represented 82.5 and 16.8%, respectively, of all the antibiotics used in Chilean aquaculture [100]. Both are broad-spectrum antibiotics used to combat infections of aquatic pathogens such as *Aeromonas salmonicida*, *Aeromonas hydrophila*, and *Yersinia ruckeri*, among others [102]. The predominance of florfenicol in recent years is mainly due to the fact that this antibiotic is the main agent against *P. salmonis*.

Pathogens cause immense economic losses that also have social impact. The crisis of the ISA virus in 2007 represented US\$ 600 million and 16,000 jobs lost. The infection by *P. salmonis* caused 70% of the mortality of Atlantic salmon and rainbow trout in recent years, amounting to US\$ 450 million per year, including vaccination, antibiotics, and other measures to mitigate the disease [103].

Although preventive measures have been implemented with the mandatory use of vaccines, the results have not been as promising as in mammals. The reason behind would be the less developed immunological memory in salmonids than mammals [104]. A corollary of this is the continued massive use of antibiotics. As mentioned, a critical problem is the propagation in the environment of microorganisms with resistance genes. Antibiotic residues have been reported in the muscles of fish and can be transferred directly to humans if the fish is not cooked properly [105]. Studies using massive sequencing to detect antibiotic resistance genes from fish sediment identified that more than 90% had mobile genetic elements of high homology to human pathogens [106]. This confirms the high rate of genetic exchange, or horizontal transmission, of antibiotic resistance between microbes and fishes that in the end affects human health.

Some studies identify the presence of antibiotic resistance genes in isolates from areas where salmonids are cultivated [107]; however, in-depth studies on the impact of antibiotics on the composition of microbial communities in farmed animals are still lacking. This field is open for metagenomics, especially for environmental DNA monitoring, in order to evaluate the impacts on the native microbial communities and their dynamics in places where salmon cages are located. It is particularly important to establish how environmental microbial communities recover after antibiotic treatment or after cages have moved to another place to allow recovery of the site, according to the actual practice.

In relation to the effect of antibiotics on the salmonid microbiota, studies conducted with culturable bacteria indicate that oxytetracycline decreases bacterial diversity, facilitating the proliferation of opportunistic pathogenic bacteria [70]. Studies conducted in our laboratory using broad-spectrum antibiotics such as bacitracin/neomycin showed the bacterial load in the intestine is reduced 10 times, in particular, the population of *Proteobacteria*, which favors the increase of the phylum *Firmicutes*. At the level of genera, the predominance of *Pseudomonas* and *Aeromonas* is replaced by *Lactococcus*. The interesting data is that the microbial composition is not recovered after 15 days of antibiotic treatment, which suggests that changes in the microbiota could be irreversible, at least within a short window of time (Tello, unpublished data). At the functional level, we were able to see that antibiotics increase the diversity of genes related to general metabolic pathways (amino acid biosynthesis, secondary metabolites, enzyme synthesis, etc.) and antibiotic metabolisms.

In farming systems, the impact of antibiotics on the fish's normal microbiota and its effects on the long term is unknown. It remains to be determined if the dysbiosis induced by the antibiotics generates a favorable scenario for other pathogens or if it affects the immune response. Preliminary work from our laboratory indicates that the administration of bacitracin and neomycin induces the expression of the inflammatory immune response judging by the increase in the expression of interleukin IL-1b and the reduced amount of leukocytes in the immunological organs. Similar effects are observed when administering florfenicol, a broad-spectrum antibiotic widely used in salmon farming (**Figure 2**).

#### **3.4 Effect of heavy metals**

It is a well-known fact that the presence of metals in different environments generates a toxic effect on both the biota and microbiota. Metagenomic analyses show

#### **Figure 2.**

*Effects of bacitracin/neomycin on the immune system of Atlantic salmon. The figure shows the effects of bacitracin/neomycin administered by 14 days on the amount of leukocytes (A) and expression of IL-1b (B). Both measures were determined on the head kidney.*

#### *Application of Metagenomics to Chilean Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.86302*

these effects are persistent in the gut microbiota and in the environment, principally in water bodies where the toxicity of these metals modulates the microbial community composition [108, 109]. Previous studies have demonstrated that environmental pollutants affect the gut microbiota even at low concentration [109]. The metagenomic analyses of the gut microbiota from *Cyprinus carpio* have made evident the prevalence (or selection) of bacterial genera containing genes associated to metal resistance as well as genes involved in heavy metal biotransformation pathways that tend to attenuate their toxicity in fishes exposed to heavy metals [110, 111].

The Chilean aquaculture industry uses heavy metals such as copper due to its antimicrobial properties in, for example, antifouling paints that avoid the formation of bacterial biofilms which also are considered potential pathogen reservoirs [112, 113]. Another application of copper is for coating cages and recirculation systems (RAS) used in land-based salmon farming sites. On a less positive side, it has been observed that small variations in the concentration of copper in incoming freshwater from underground wells that feed a hatchery modify the fish behavior and reduce food intake, such as in cascade effect that ends up affecting the production. Such behavior impairment by copper seems to affect the nervous system of the fish [114]. Other studies in *Cyprinus carpio* show that low copper concentrations also affect the microbiota diversity and lipid metabolism [110].

Owing to the importance of recirculation systems in aquaculture facilities, it is imperative to understand how environmental pollutants affect the dynamic of the gut microbiota and health of reared fishes as well as the microbiota of the biofilter [115]. The metagenomic approach combined with metabolomic studies may help to understand the complex changes that occur at different levels in a hatchery exposed to environmental pollutants.
