opened for admissions at May 18, 2018. The new hospital consisted of 522 single-occupancy rooms with private bathrooms. This prospective cohort study included patients admitted from May 18, 2018 until September 1, 2019. Adult patients admitted to departments cardiology, gastroenterology and hepatology, general surgery, hematology, internal medicine, nephrology, neurology, neurosurgery, orthopedics, or plastic surgery with an expected stay of more than 48 h were asked to participate at admission. Patients with multiple hospitalizations during the study period were allowed to participate more than once. Participating patients received a questionnaire with accompanying return envelope, and a perianal swab (flocked swab [ESwab Copan Italia, Brescia, Italy] was obtained within 24 h of admission and transported in its accompanying 1mL Amies medium). Samples were taken by trained members of the research team, or patients could self-sample with instructions from the members of the research team. Questionnaire A questionnaire and a patient information form were designed in Dutch (see Additional file 1). The questionnaire was pilot tested on three persons and adjusted accordingly. The questionnaire included questions about risk perception (i.e. awareness and feelings about international travel and risk of acquiring HRMO), contact with domestic and farm animals, antibiotic use <1 year, antacid use <1 year, travel history <1 year of persons living in the same household, and travel history of the patient <1 year. If patients did travel, questions were asked about behavior during travel (e.g. pastry and ice cream consumption), use of malaria prophylaxis, experiencing travelers’ diarrhea and/or vomiting, hospitalization, antibiotic use, and antacid use during travel. Microbiological methods Samples collected from May 18, 2018, until January 19, 2019, were stored in a -80°C freezer before being processed. To prevent freezing/defrosting damage, 0.2mL 99% glycerol was added to the samples before freezing. Samples taken after January 19, 2019 were processed directly. All samples, regardless of being frozen, were processed using the same procedure. Samples were screened for highly resistant Pseudomonas aeruginosa, -Acinetobacter baumannii, -Enterococcus faecium, and -Enterobacterales. First, 250µL was placed in an Enterococcosel Broth (BD diagnostics, Sparks, USA) with amoxicillin 8mg/L and incubated overnight at 35°C. From this broth, a Vancomycin Screen Agar (VSA, BD diagnostics, Sparks, USA) plate was inoculated and incubated twice overnight at 35°C. Second, 250µL was placed in a tryptic soy broth with vancomycin (50mg/L) and incubated overnight at 35°C. From the vancomycin broth, a ChromID Carba Smart plate (bioMérieux, Marcy l’Etoile, France) was inoculated on both sides and incubated overnight twice at 35°C. Additionally, from the vancomycin broth, a BrillianceTM ESBL Agar (Oxoid, Basingstoke, UK) was inoculated and incubated twice overnight at 35°C. For all plates, colonies were identified using MALDI-TOF MS (Bruker Daltonik, Bremen, Germany). In case of P. aeruginosa, isolates were tested for the presence of blaOXA-48, blaKPC, blaIMP, blaVIM, blaNDM genes, using PCR, with use of established procedures. When negative, a Carbapenem Inactivation Method (CIM) test was performed (6). For A. baumannii isolates and for ESBL suspected colonies, antibiotic 54 Chapter 2.2
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