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Background and Aim: In the last few decades co-trimoxazole, an antibacterial combination of trimethoprim and sulfamethoxazole, has been used for treatment of bacterial infections, but due to the vast usage of these drugs, resistant strains have appeared throughout the world. One of the reasons for resistance to co-trimoxazole is related to drf genes, which are responsible for trimethoprim resistance, while the sulfamethoxazole resistance is due to sulfonamide sul genes. The aim of this study was to investigate drug resistance and frequencies of resistance genes in gram-negative bacteria isolated from clinical specimens. Materials and Methods: Clinical samples were collected from patients in Pars Hospital, Tehran, Iran, in which presence of gram-negative bacteria was confirmed by biochemical tests. Then antibiotic susceptibility tests were performed for 5 antibiotics by disk diffusion agar technic. DNA was extracted from bacteria resistant to co-trimoxazole, followed by PCR using specific primers for sul1, sul2, sul3, and drf7 genes. Results: In the co-trimoxazole-resistant bacteria, 26%, 74%, 2% and 16% of the isolates contained sul1 gene, sul2 gene, sul3 gene and drf7 gene, respectively. Further analysis of the data showed that 51% of the isolates were resistant to gentamicin, 74% to ceftriaxone, 65% to ciprofloxacin and 3% to colistin. For resistance to trimethoprim only the drf 7 gene was used. Conclusion: The results of this study show that in the isolates of co-trimoxazole-resistant gram-negative bacteria, the sul 2 gene has a major role in development of resistance to sulfonamides. In this study only the drf 7 gene was used to assess the resistance of trimethoprim, so we recommend to conduct studies also on other drf genes, so that the importance of each in resistance to co-trimoxazole can be determined. [ABSTRACT FROM AUTHOR]