Supplement 2. Microbiological methods and whole genome sequencing Microbiological methods Risk based screening samples For methicillin-resistant Staphylococcus aureus (MRSA), nose, throat, and perineal samples were taken using cotton swabs (Copan Italia, Brescia, Italy). For vancomycin-resistant Enterococcus faecium (VRE), a rectal swab was taken. For the Gram-negative antibioticresistant bacteria, throat and rectal samples were taken, also with cotton swabs. To determine the presence of MRSA, the swab was placed in a tryptic soy broth (TSB) with 6.5% NaCl and incubated for 24 h at 35°C. Subsequently, 10 µl of broth was subcultured on a BBL-CHROMagar MRSA (BD diagnostics, Sparks, USA) which was incubated for 48 h at 35°C. Plates were checked at 24 h and 48 h. Selected pink and purple colonies were identified with the Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF) (Bruker Daltonics, Bremen, Germany). A suspension of 0.5 McFarland was made from S. aureus isolates, and used to perform a cefoxitin disk diffusion (30 µg; Oxoid, Basingstoke, UK) on a Mueller Hinton agar (BD diagnostics, Sparks, USA). A growth inhibition zone of <22mm after 18 to 24 h was considered resistant. To confirm presence of MRSA, a mecA/mecC PCR was performed, using established procedures. To determine the presence of VRE, the swab was placed in an Enterococcosel broth (BD diagnostics, Sparks, USA) with 8 mg/L amoxicillin and incubated overnight at 35°C. From the broth, a BrillianceTM VRE (Oxoid, Basingstoke, UK) was inoculated and incubated twice overnight at 35°C. Selected blue/purple colonies were identified using the MALDI-TOF and antibiotic susceptibility was determined with the VITEK®2 (bioMérieux, Marcy l’Etoile, France). For E. faecium colonies resistant for amoxicillin, an Etest for vancomycin, an Etest for teicoplanin, and a vanA/vanB PCR using established procedures, were performed to confirm the presence of VRE. To determine the presence of extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E), a BrillianceTM ESBL agar (Oxoid, Basingstoke, UK) was inoculated and incubated twice overnight at 35°C. To determine the presence of carbapenemaseproducing Gram-negative bacteria, a ChromID CarbaSmart agar (bioMérieux, Marcy l’Etoile, France) was inoculated and incubated twice overnight at 35°C. All colonies were identified using the MALDI-TOF and antibiotic susceptibility was determined with the VITEK®2. ESBL production was confirmed phenotypically, using double disk diffusion test. Carbapenemase production was tested with the carbapenem inactivation method (CIM) test, presence of carbapenemase genes with a multiplex PCR for blaVIM, blaIMP, blaNDM, blaKPC and blaOXA-48-like (1). To determine the presence of blaOXA-48-positive Gram-negative rods, the swab was placed in a TSB with 0.25 mg/L ertapenem and 50 mg/L vancomycin and incubated overnight at 35°C. From the broth, a PCR for blaOXA-48 was performed. In case of a positive PCR result, the broth was subcultured on a ChromID CarbaSmart agar and a MacConkey agar (Biomerieux, 92 Chapter 2.3
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