Background: E. coli may develop different complex mechanisms of resistance to various classes of antibiotics. Objectives: To assess the prevalence of E. coli in Menoufia University Hospitals, to evaluate its antibiotic resistance profile and to detect the presence of aminoglycoside resistance genes [aac(3)IIa and aac(6)Ib] by real-time polymerase chain reaction (PCR). Methodology: A total of 200 clinical samples were collected from patients in Menoufia University Hospitals. E. coli was identified by standard microbiological methods. E. coli isolates were tested by the modified Kirby-Bauer disk diffusion method, and for extended-spectrum β-lactamases production. Aac(3)IIa and aac(6)Ib genes were detected by real-time PCR. Results: E. coli forms 25% of the isolated bacterial strains. E. coli isolates showed the highest resistance to pipracillin and amoxicillin-clavulanate (96%). On the other hand, carbapenemes had the least resistance rate (30%) followed by amikacin (44%), gentamicin (60%),
tobramycin (60%) and streptomycin (64%). ESβL production was found among 70% of the isolates by the screening and 50% by the confirmatory methods. Real-time PCR showed that 84% of the isolates contain aac(3)IIa, 52% contain aac(6)Ib and 46% contain both genes. Conclusion: E. coli showed resistance to different classes of antibiotics including aminoglycosides with high prevalence of aac(3)IIa and aac(6)Ib genes. Therefore, infection control measures and rationalized use of antibiotics are mandatory.