## 3 Results and Discussion
### Phenotypic Characterization of Antibiotic Resistance  
The _Enterobacteriaceae_ isolates obtained from the samples collected for three consecutive years showed high resistance to most of the ABs tested (Figure 4). Among the \(\beta\)-lactams, penicillin (amoxicillin/ clavulanic acid), 2 nd generation cephalosporporins (FOX), 3 rd generation cephalosporporins (CAZ and CTX) and 4 th generation (CPO) were the ABs for which more resistances were found. Regarding the non-\(\beta\)-lactam AB classes, a higher percentage of isolates resistant to tetracycline and sulphonamides AB (TET and SXT, respectively) was observed. This result can be explained by the intensive use of this class of AB for many years in veterinary medicine and intensive animal production in the region [4]. The AB resistance patterns of samples from the inlet of Block II showed 100% resistance to AMC, FOX, ATM and TET, but lower resistance to the other ABs tested (Figure 4a). Soil water samples from the paddies, in addition to those resistances, showed 100% resistance to CTX, CHL and SXT, which had already been detected in the irrigation water, although less prevalently (Figure 4b,c). The highest resistances were detected at the outlet of Block II (Figure 4e). Figure 4: Relative frequency of antibiotic resistance patterns of _Enterobacteriaceae_ isolates obtained from samples collected in 2019, 2020 and 2021 from: (**a**) Block II Inlet; (**b**) Paddies’ Inlet; (**c**) Rice paddies—soil water; (**d**) Paddies’ Outlet; (**e**) Block II Outlet; (**f**) LVID Outlet. AB: Nine \(\beta\)-lactams: ampicillin (AMP), amoxicillin/ clavulanic acid (AMC), ceftazidine (CAZ), cefotaxime (CTX), ceftprone (CPO), azretonam (ATM), cefoxitin (FOX), imipenem (IPM), meropenem (MEM) and five **b**: chloramphenicol (CHL), tetracycline (TET), gentamicin (GEN), trimethoprim/ sulfamethoxazole (SXT) and ciprofloxacin (CIP). The higher percentages of resistances to ABs detected in drainage water samples (Figure 4d,e), compared with irrigation water and paddies' samples, might be explained by the leaching of native microorganisms from the soil and water that have been accumulating resistances due to the cultural practices used in this hydraulic block, namely direct grazing in forage crops, and the soil incorporation of manure to some other non-rice-crops, for which drainages are all collected in the Rio Negro ditch (Block II Outlet). Therefore, this sampling site represents the resistance determinants that occur in the entire Block II, because the Rio Negro ditch works as a collector for all of them. The isolates from the paddies showed higher relative resistance percentages for both \(\beta\)-lactams and non-\(\beta\)-lactams than those from the irrigation water at the Block inlet, except for CIP, at which no resistances were detected. At the outlet of the Block, these values are even higher. An explanatory hypothesis for these results lies in the fact that, as drainage water is reused for irrigation, it enters a cycle that allows the accumulation of resistant microorganisms, thus favoring the transfer of AB resistance genes by bacterial recombination. The increase in the relative variation frequencies of AB resistances in soil water of the paddies (Table 3) is also possibly due to the conditions of the rice production ecosystem, facilitating AB resistance gene transference. There is also a high resistance to \(\beta\)-lactams for the isolates from the samples collected at the paddy inlet and outlet and at the Block II outlet (Figure 4b,d,e), which represents surface water. This high prevalence of isolates resistant to \(\beta\)-lactams, especially to penicillin and 2 nd, 3 rd and 4 th generation cephalospors is probably due to the use and abuse of this class of ABs for many years in livestock for the treatment of infections and as a growth promoter. The livestock production in the area surrounding the LYID possibly explains these high AB resistance frequencies.