Introduction Highly resistant microorganisms (HRMO) are a common cause of healthcare-associated infections (HAI), and are a worldwide threat to public health and modern healthcare (1). Among HRMO, extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E) are most frequently identified. Worldwide, the prevalence of ESBL-E in the community differs from 2% to 46% (2). In hospitals, this prevalence is higher and outbreaks with ESBLE occur. Hospital design is thought to play an essential role in the spread of HRMO including ESBL-E (3-5). To decrease the spread of HRMO within hospitals, the Facility Guideline Institute recommends transitioning to 100% single-occupancy rooms for medical/surgical units (6). Moreover, their 2018 report advises 100% single patient rooms in adult critical care units (7). An added benefit of single-occupancy rooms is that they remove the necessity for intra-hospital patient transfers for small procedures, social circumstances (e.g. end-oflife care), or for an indication of contact isolation (8). By reducing the number of intrahospital patient transfers, which leads to less exposure of the patient to different hospital environments, and by reducing the exposure to unidentified infected or colonized roommates, the implementation of 100% single-occupancy rooms is expected to reduce the risk of HRMO acquisition and transmission (9). However, current literature shows conflicting results for the effect of single-occupancy rooms on the acquisition of HRMO (4, 10, 11). Furthermore, literature on the effect of single-occupancy rooms on ESBL-E acquisition is limited to the comparison of ESBL-E acquisition between an intensive care unit (ICU) with an open plan and an ICU with single-occupancy rooms, which showed no significant difference (11). In May 2018, the Erasmus MC University Medical Center (Erasmus MC) relocated from an old hospital building, with mainly multiple-occupancy rooms, to a newly constructed hospital building with 100% single-occupancy rooms. We used this unique opportunity to determine the effect of relocating to a new hospital with 100% single-occupancy rooms on the acquisition of ESBL-E by determining ESBL-E carriage in patients at admission and discharge in both buildings. Whole genome sequencing (WGS) was used to determine if strains at discharge were identical to those present at admission or the result of acquisition during hospitalization. Additionally, we aimed to determine the effect of intra-hospital patient transfers on ESBL-E acquisition, and to identify the percentage of ESBL-E carriers that remained undetected by clinical samples. Methods Study design and setting This study was performed at the Erasmus MC, a university medical center located in Rotterdam, the Netherlands. On May 18, 2018, the adult clinic of the Erasmus MC relocated from an old, 1200-bed hospital building with mainly multiple-occupancy rooms and shared bathrooms, to a newly constructed 522-bed hospital building with 100% single-occupancy rooms and private bathrooms. To determine the prevalence of colonization with ESBL-E and the incidence of acquisition of ESBL-E in the old and new hospital building, a prospective before-and-after study was performed. Participating departments were cardiology, 2 27 Effect of single-occupancy rooms on ESBL-E acquisition
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