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IntroductionNosocomial pathogens are responsible for severe infections and are typically acquired in healthcare settings within days of patient admission. One of the primary contributors to the spread of healthcare-associated infections (HAIs) is the transfer of bacteria from inanimate surfaces to patients.MethodsA cross-sectional study was done at Zewditu Memorial Hospital from June to November 2024. For this study, 204 inanimate surfaces from operating rooms and intensive care units were swabbed and cultured on MacConkey and blood agar. Bacteria were identified based on their colony morphology, gram staining, and conventional biochemical tests. Antimicrobial susceptibility testing was done using disk diffusion. Extended-spectrum beta-lactamase (ESBL) producing gram-negative bacteria were confirmed phenotypically using the double disc synergy and combination disc methods, while carbapenemase producers were identified using the Modified Carbapenem inactivation method. Methicillin-resistant Staphylococcus aureus (MRSA) was detected via the Cefoxitin Disk Diffusion Test. A P-value of less than 0.05 was considered statistically significant. Data were analyzed using SPSS version 20.ResultsOf the 204 swabbed samples, 77.45% (n = 158/204) showed bacterial growth, yielding a total of 171 bacterial isolates. Among these, Gram-positive bacteria comprised 71.3% (n = 122/171), while Gram-negative bacteria accounted for 28.7% (n = 49/171). The most prevalent isolates were Coagulase-negative Staphylococcus (CoNS), making up 46.1% (n = 79/171), followed by Bacillus spp. at 21.6% (n = 37/171). Out of the total isolates, 55 were identified as pathogenic bacteria based on their potential to cause disease and selected for antibiotic resistance testing. P. aeruginosa 36.7% (n = 18/49), Acinetobacter spp. 16.3% (n = 8/49), E. coli 14.2% (n = 7/49) and S. aureus 4.9% (n = 6/122) were the commonest pathogenic bacteria identified. Gram-negative bacteria showed high resistance to ampicillin (67.3%), amoxicillin and clavulanic acid (61.2%), ciprofloxacin (63.2%), sulfamethoxazole-trimethoprim (63.2%), cefepime (57.1%), and piperacillin-tazobactam (55.1%). Similarly gram-positive bacteria showed high resistance to azithromycin (100%), penicillin (100%), clindamycin (100%), and erythromycin (100%). Multidrug resistance was observed in 61.8% (n = 34/55) of the tested gram-negative and gram-positive isolates. The incidence of ESBL-producing and carbapenemase-producing bacteria among the suspected gram-negative bacteria was 26.5% (n=13/49) and 12.2% (n=6/49), respectively. The prevalence of MRSA was 50% (n=3/6).ConclusionThe study identified a significant presence of multidrug-resistant bacteria in the hospital environment and on inanimate surfaces, emphasizing the urgent need for effective infection prevention and control measures.