## 4 Discussion
Toxins such as cytotoxic necrotizing factor 1, autotransporter toxins, and alpha-hemolysin enhance _E. coli_ virulence by targeting the cell's structure, metabolism, or cytoplasmic membrane [74]. Each pathotype exhibits a distinct virulence profile determined by gene clusters located in the chromosome or mobile elements encoding specific proteins [75]. Table 4 illustrates the virulence traits of _E. coli_ O157 strains. A comparison between sorbitol-negative and sorbitol-positive strains suggests that sorbitol-positive strains are slightly more virulent. In this category, all strains produce both toxins, and more strains demonstrate biofilm capacity and exhibit ESBL resistance compared tosorbitol-negative strains. Table 3 depicts the virulence traits of non-O157 _E. coli_ isolates, indicating they are generally less virulent than O157 isolates. There are notable differences in virulent metabolic traits between O157 and non-O157 isolates. All O157 isolates, regardless of sorbitol reaction, produce amylase and lecithinase, test positive for gelatinase, and exhibit serum resistance. However, among non-O157 isolates, the presence of these traits varies among groups. It is important to note that not all non-O157 isolates are less pathogenic, as many possess all aforementioned virulence traits. Biofilm formation is a significant virulent trait in discussions concerning _E. coli_, particularly due to the \"restaurant hypothesis\". According to this theory, commensal _E. coli_ colonizes the intestine as part of a biofilm microbiome, absorbing essential nutrients through cross-feeding from anaerobes that decompose polysaccharides. Interestingly, they also exhibit antagonistic behavior against individual planktonic _E. coli_ cells [76; 77; 78]. In our study (Table 7), 52.17% of the strains, regardless of their \"O\" classification, demonstrated biofilm-forming abilities, with almost half of these strains exhibiting this capacity to the highest degree. The survival of such strains in the C groups (46.74%) can be attributed to the increased protection provided by biofilms, even against antibiotics [79; 80; 81; 82]. Mastitis treatment in our study was administered by professional veterinarians. Empirical selection of antibiotics at the farm level was the norm, meaning all animals affected by mastitis received the same antibiotic based on the farm's therapeutic history. Antibiotic susceptibility testing or other laboratory diagnostic methods were often avoided due to increased treatment costs, leading veterinarians to rotate antibiotics frequently to prevent the emergence of resistant strains. Farmers and farm personnel were experienced in recognizing clinical mastitis early and promptly calling veterinarians. They were also well informed about the importance of withdrawal periods, ensuring compliance to prevent milk rejection. Our findings (Table 5) demonstrate varying resistance frequencies depending on the antibiotic class used, an expected observation, along with some unexpected discoveries. The twelve antibacterial substances utilized in this study belonged to five distinct classes based on their structure and mode of action. Group C1, receiving a combination of procainic benzylpenicillin and dihydrostreptomycinomycin, showed less impact on \(\beta\)-lactam resistance, with increased resistance observed in strains against amoxicillin, which is structurally and functionally similar to procainic benzylpenicillin. Resistance to Cephalothin (CEP), a third-generation \(\beta\)-lactam, was less prevalent, while resistance to cefotaxime, a fourth-generation \(\beta\)-lactam, remained unaffected. It appears that as \(\beta\)-lactams evolve, resistance mechanisms that were effective against older members of the family have a diminishing or negligible impact on newer members. The increased resistance frequencies to both streptomycin and chloramphenicol may be attributed to a mechanism of cross-resistance, as these antibiotics have different structures but similar modes of action (interfering with the 70s ribosomal subunit).