Knowledge, Practices, and Attitudes Regarding Antimicrobial Resistance and Antibiotic Use Among Hospital Staff: Findings from a Two-Round Cross-Sectional Survey in an Italian Tertiary Care Hospital

Background: Education is a cornerstone of antimicrobial stewardship, yet evidence on its impact across the entire hospital workforce remains limited. This study aimed to assess knowledge, attitudes, and practices regarding antibiotic use and AMR among workers employed at an Italian tertiary-care hospital, and to explore the association between questionnaire performance and participation in antibiotic-related training activities. Methods: A repeated cross-sectional survey was conducted in 2024 and 2025 at a large Italian tertiary-care hospital (Padua, Northern Italy). A structured questionnaire adapted from an ECDC-validated instrument was distributed through the institutional email system to all hospital employees, including healthcare professionals and non-healthcare workers. The questionnaire investigated demographic and professional characteristics, participation in antibiotic-related training activities, and knowledge, attitudes and practices regarding antibiotic use and AMR. Questionnaire performance was assessed using both a binary scoring system and a partial credit scoring system. Multivariable linear regression analysis was performed to identify independent predictors of performance. Results: A total of 3193 participants completed the survey (1928 and 1265 in 2024 and in 2025, respectively). The mean (SD) total score was 5.75 ± 2.25 out of 15 points, with the lowest performance observed in the practice domain. No significant differences were found between the two survey years (2024 and 2025). Participants who attended antibiotic-related training performed significantly better than untrained participants (6.23 vs. 5.01; p < 0.001), with prior training emerging as the strongest independent predictor of questionnaire performance (β = 4.8; p < 0.001). Younger age was associated with better performance, while longer seniority (>30 years) was associated with lower scores. Among healthcare professionals, modestly higher scores were observed in 2025 compared with 2024, particularly in the practice domain. Conclusions: Persistent opportunities for improvement remain in AMR-related knowledge, attitudes and particularly practices across the hospital workforce. Participation in antibiotic-related training activities was associated with improved questionnaire performance, supporting the role of structured and continuous educational interventions within hospital AMS programs. Repeated institutional educational initiatives may contribute to strengthening stewardship-related awareness and practices over time.