The E. coli H7:O157 was the subject of many studies. It is characterized by producing Shiga toxins, abdominal illness, and developing resistance to antimicrobial agents. A total 20 fecal samples tested positive for E. coli H7:O157 from patients showed the typical symptoms of the infection, and another 20 from animals were collected. The bacterium was isolated, identified, and classified using culture medium, and molecular methods. Results obtained from classification of E. coli H7:O157 showed it is highly similar to E. coli H7:O157 strain Sakai. The genes rpoB, stx, waa, and rfbO were deposited in the NCBI website under accession no. PP059841, OR939814, PP059843, and PP059842 respectively. The mutant sequences of waa sites K, L, Y were analyzed to determine the shifting in their function, cell wall formation, and the ability to develop antibiotic resistance the mutant E. coli H7:O157 compared to the wild type. We found that the resistance to antibiotics in mutant E. coli H7:O157 increased significantly when compared to the wild type. Animals. The explanation for this may be attributed to the waa K, and waa L by preventing theentry of antimicrobial agents into the bacterial cell.