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Background and Objectives: Due to the reduced susceptibility of clinical Clostridioides difficile strains in hospitals to various antimicrobial agents, the importance of antimicrobial susceptibility testing (ASTs) has increased. This study aimed to investigate the toxin gene profiles and the antimicrobial resistance of C. difficile isolated from hospitalized patients suspected of having Clostridioides difficile infection (CDI) in Tehran, Iran. Materials and Methods: The stool samples were obtained from a hospitalized patients. The samples were shocked by alcohol and the patients cultured on cycloserine-cefoxitin-fructose agar in anaerobic Conditions. Toxin assay was performed for detection of toxinogenic isolates. An antibiotic susceptibility test was done. Furthermore, their genome was extracted for PCR to confirm C. difficile and detect toxin gene profile. Results: Toxigenic C. difficile were identified in 21 of the 185 stool samples (11.3%). PCR detected seven toxin gene profiles; the highest prevalence was related to tcdA+B+, cdtA+B- toxin gene profile (57.1%). There were 14.3% and 28.6% resistant rates of the isolates towards vancomycin and metronidazole with the toxin gene profiles; tcdA+B+, cdtA±B+; and tcdA+ B-, cdtA-B+. All resistant isolates to moxifloxacin, clindamycin, and tetracycline were belonged to the toxin gene profiles; tcdA+B+, cdtA+B+; tcdA+B+, cdtA+B-, and tcdA-B+, cdtA+B-. Conclusion: Relative high resistance was detected towards metronidazole and vancomycin, although, still have acceptable activity for CDI treatment. However, a proper plan for the use of antibiotics and more regular screening of C. difficile antibiotic resistance seems necessary. [ABSTRACT FROM AUTHOR] more...