Background <p>The spread of multidrug-resistant microorganisms (MDROs), including extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E), has increased worldwide and constitutes a significant public health challenge. International guidelines vary in their recommendations for isolation in single rooms and contact precautions regarding carriers of MDR microorganisms to mitigate cross-contamination in the ICU. The aim of this study was to investigate whether contact precautions without single-room isolation prevent cross-contamination of ESBL-E in an intensive care unit (ICU).</p> Methods <p>All patients admitted to a general ICU during a period of 19 months were included. The study began before the COVID-19 pandemic and continued, albeit interrupted during the first wave, through the second and third wave. Rectal swabs, swabs from drainages and intravenous catheters were sampled for the detection of ESBL-E in all patients at the time of admission. Swabs were also taken from all patients co-treated with an ESBL-E-positive patient (i.e., the index patient) at the time of discharge. All cross-contaminated patient bacterial isolates were analyzed with whole-genome sequencing and compared to the isolate from the corresponding index patient.</p> Results <p>Of 1042 patients admitted to the ICU, 82 patients were index patients, either known ESBL-carriers or tested positive at admission. 365 ESBL-E-negative patients (n=365) at ICU admission were co-treated in the same room as an index patient during their ICU-stay. Post-ICU discharge, three patients from the latter group tested positive for ESBL-E. No bacterial ESBLisolates from the latter patients corresponded to those of the index patients when their bacterial genomes were identified and compared.</p> Conclusions <p>Contact precautions without single-room isolation of ESBL-E-positive patients did not result in any cross-contamination between ICU-patients in an endemic setting with a short length of stay.</p>