**5. Microbial resistance**

Genotoxic and enzymatic effects on aquatic organisms exposed to ATBs were also observed by several authors. For example, Botelho et al. (submitted manuscript) reported genotoxic effects of oxytetracycline and florfenicol ATBs in concentrations found in the water of a major Brazilian reservoir where fish farming activity is practiced with *Oreochromis niloticus* fish species. In this study, DNA damage was observed using the comet test when exposed to

Oliveira et al. [98] observed the inhibition of catalase activity in adult brain and gills of *Danio rerio* fish when exposed to higher concentrations of amoxicillin (50 and 100 mg L-1). There was also a tendency for the induction of glutathione S-transferase (GST) enzyme at all concentra‐ tions of the same ATB. In this same study, a dose-dependent catalase was observed in the brain of *D. rerio* adults after oxytetracycline exposure, while GST activity increased after exposure

Most of the studies related to ATBs effects on aquatic organisms refer to acute effects (mainly lethality) in a short period of time. Note that in the aquatic environment, due to the phenom‐ enon of dilution, the concentrations of chemicals in general, including ATBs, are found at the levels of µg L-1 and ng-1. Thus, the observed effects will be chronic, i.e., at a considerably longer period than that observed for acute effects. Thus, in toxicity evaluations, especially to aquatic organisms, the use of environmentally relevant concentrations should be taken into account since this way the effects will be more realistic and will portray in a more real way what

Soil plays important roles in ecosystems since it is the basis of nutrients and the animal and plants habitat, in addition to functioning as an immense bioreactor, where the degradation of pollutants and nutrients transformation occurs. However, as already seen in this chapter, the soil may also be the final destination of ATBs used in veterinary medicine originating from manure and sewage mud used to fertilize vegetables [99] or from package disposal. Due to the ecological importance of soil for the ecosystem, it is important to know whether or not ATBs

As shown earlier in this chapter, once in the soil, depending on the physical and chemical characteristics of the ATBs and the soil, they may follow different pathways, such as being leached or carried superficially by rain, contaminating aquatic environments (low *K*d values)

In general, the effects of ATBs to aquatic organisms are higher than those of soil fauna, and thus little is known about the toxicity of these drugs for these organisms. According to Ding and He [100], once in the soil, ATBs can change the structure of the microbial community because even to those which have a broad spectrum of action, selective effects on several microorganism groups may occur. As a result, the relative abundance of microorganisms is

The sorption of pollutants in general in the soil is one of the major mechanisms controlling toxicity, by reducing its availability [101]. Thus, in toxicity studies with chemical agents, the choice of a molecule with low *K*d is recommended. In addition, toxicity to organisms in the soil decreases over time due to transformations the molecule undergoes over time through less

concentrations of 425 and 4000 ng L-1 of florfenicol and oxytetracycline, respectively.

to concentrations higher than 1 mg L-1 of oxytetracycline in muscle and liver samples.

happens in the environment if such chemical agents are present.

124 Emerging Pollutants in the Environment - Current and Further Implications

changed, interfering with the interactions between different species.

have negative effects on the fauna.

or persisting in the soil (high *K*d values).

One of the biggest problems related to the use of ATBs, in addition to those already discussed in this chapter, is the development of bacterial strains resistant to ATBs in the environment, mainly due to the continuing use of these drugs at low concentrations. Bacterial resistance arises and is maintained by mutations in the bacterial DNA or by horizontal gene transfer mechanisms, which include conjugation with other bacteria, transduction with the bacterio‐ phage, and free DNA uptake via transformation.59 In the case of continuous and prolonged use of sublethal concentrations and the subsequent elimination of feces in the soil, they could cause the sharing of resistant plasmids to nonresistant organisms [18, 99]. Another possibility of occurrence of bacterial resistance is that low concentrations of ATB residues transferred to the soil by the application of contaminated animal manure favor the selection of resistant populations [109]. However, the direct introduction of resistant microorganisms derived from feces of animals treated with ATBs seems to be more important to resistance [99] than induction due to the presence of ATB residues on the environment.

It is important to remember that there is a large reservoir of ATBs-resistant bacterial genes in the soil. However, according to Schmidt e al. [110], it is not known whether this occurs naturally or due to the use of veterinary ATBs. As an example, in a study by Esiobu et al. [102], isolated bacteria of a garden soil fertilized with dairy cattle manure showed 70% resistance to ampi‐ cillin, penicillin, tetracycline, vancomycin, and streptomycin ATBs.

The exposure intensity of bacteria to ATBs agents influences the amplitude of its resistance, and the exposure intensity usually depends on the origin of the treatments by which bacteria were submitted. Costanzo et al. [111] indicated that bacteria from a sewage treatment plant reactor were resistant to ciprofloxacin, tetracycline, ampicillin, trimethoprim, erythromycin, and sulfamethoxazole antibiotics, while bacteria isolated from the effluent receiver water‐ course showed resistance to erythromycin and ampicillin. This same study showed that erythromycin, clarithromycin, and amoxicillin ATBs, at a concentration of 1.000 µg L-1, decreased more significantly the rate of bacterial denitrifying.

In aquaculture, the intensive use of ATBs provides a selective pressure for the creation of bacteria resistant to drugs and genes resistant to transmitted pathogens of fish and other bacteria in the aquatic environment. From these resistant bacteria, resistance genes can be spread by horizontal gene transfer and transfer to human pathogens. Drug-resistant pathogens present in the aquatic environment can directly reach humans. The horizontal gene transfer can occur in the aquaculture environment, in the food chain, or in the human intestinal tract. Among ATBs commonly used in aquaculture, several are classified by the World Health Organization (WHO) as extremely important for use in humans. The occurrence of ATBs resistance in human pathogens severely limits the therapeutic options in human infections. Taking into account the rapid growth and the importance of the aquaculture sector in many regions of the world, due to the widespread, intense and often irregular use of ATBs in this animal production area, efforts are necessary to prevent the development and spread of bacterial resistance in order to reduce the risk to human health [112].

Another issue in aquaculture regarding bacterial resistance needs to be highlighted, that is, if bacterial populations are resistant to a certain ATB used in this sector, or the producer changes the ATB or increases the dose in anticipation of a more efficient control. However, these two practices make such microorganisms to become resistant to this new applied molecule over time. Increasing the dose may also have negative effects on native aquatic biota of where the creation is installed.

In the study of Akinbowale et al. [113], isolated bacteria from water samples and organisms used in aquaculture showed widespread resistance to ampicillin, amoxicillin, cephalexin, and erythromycin ATBs; frequent resistance to oxytetracycline, tetracycline, nalidixic acid, and representatives of the sulfonamide group; and infrequent resistance to florfenicol, chloram‐ phenicol, ceftiofur, oxolinic acid, gentamicin, and trimethoprim. In another study performed on the Ilha Solteira Reservoir, São Paulo, where one of the largest and most important aquaculture parks in Brazil is located and where fish farming is intense, Monteiro [46] studied the bacterial resistance in Nile tilapia kidneys, which is a species cultivated in this place, and observed bacterial resistance to sulfonamides, quinolones, and tetracyclines. These two examples from cited studies confirm that both aquaculture products and water from aquacul‐ ture environments have risks of transferring ATBs-resistant bacteria to humans through these product consumption and contact with water, as mentioned in the previous paragraph. Remember that in the case of the Ilha Solteira Reservoir, this environment is an important aquaculture redoubt where the population uses its waters for water sports, in addition to fishing and fish consumption.

As a direct consequence of bacterial resistance, there is the increased frequency of ineffective treatments, increased severity of infections, prolonged duration of diseases, increased fre‐ quency of bloodstream infection, increased hospitalization, and increased mortality. The prolongation of diseases has been demonstrated in case-control studies of Campylobacter resistant to fluoroquinolones, and the increased severity of infections of *Salmonella typhimuri‐ um* resistant to quinolones was also demonstrated, as well as increased morbidity or mortality also assigned to nontyphoidal *Salmonella* serotypes and to Campylobacter [112].

Several studies have reported the occurrence of bacterial resistance in environmental com‐ partments, such as in wastewater, groundwater, surface water, sediments, and soils [114–119].

As discussed so far, bacterial resistance is a threat to the effectiveness of ATBs in animal husbandry and to the health of the environment. Therefore, the prudent use of these molecules in all livestock sectors seems to be the solution to combat or reduce this problem.
