taken, with the exception of the bottom of the sink plug. Since it was not feasible to sample the bottom of the sink plug with a RODAC, CFU counts were not determined for this location. The RODACs were pressed firmly on surfaces for about 10 s, according to standard practice. For the door handle and the top of the sink plug, the RODAC was carefully rotated over the surface, to ensure that the whole RODAC came in contact with the surface. Sterile cotton swabs (BSN medical, Almere, the Netherlands) were used to determine the presence of MRSA, vancomycin-resistant Enterococcus faecium (VRE), extended-spectrum βlactamase (ESBL)-producing Enterobacterales (ESBL-E), carbapenemase-producing Enterobacterales (CPE), carbapenemase-producing Pseudomonas aeruginosa (CP-PA), and carbapenem-resistant Acinetobacter baumannii (CR-AB). For each sampling site, two swabs were pre-wetted with phosphate buffered saline (PBS) before sampling a standardized surface of 100cm2 (Supplementary file 2). During sampling, swabs were rotated and moved in multiple directions as predefined in our sampling protocol (Supplementary file 2). Due to the specific shapes of door handles, shower drains and the top and bottom of the sink plug, no standardized surface of 100cm2 was sampled. Instead, the complete surfaces were sampled, while the swab was rotated and moved in multiple directions according to our protocol. Directly after sampling, in random order, one swab was placed in a tryptic soy broth (TSB) with aztreonam 75 mg/L (aztreonam broth) and one swab in TSB with vancomycin 50 mg/L (vancomycin broth). Microbiological methods RODACs were incubated twice overnight at 35°C, after which CFUs were counted. When more than 100 colonies were counted, this was reported as >100 CFU. Both the vancomycin and the aztreonam broth were incubated for 24 hours at 35°C. On the incubated aztreonam broth, a vanA, vanB, mecA/mecC PCR was performed using established procedures. When the vanA/B PCR was positive, a BrillianceTM VRE (Oxoid, Basingstoke, UK), was inoculated and incubated twice overnight at 35ºC. All suspected Enterococcus spp. colonies were identified to species level using Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF) (Bruker Daltonics, Bremen, Germany) running the MBT Compass Library, Revision E; MBT 7854 MSP Library and MBT Compass Library, Revision F MBT 8468 MSP Library. For E. faecium isolates, an additional vanA and vanB PCR was performed. When the mecA/mecC PCR was positive, a TSA plate with 5% sheep blood (blood agar [Becton Dickinson, New Jersey, USA]) and a BBLTM CHROMagar TM MRSA II* (Becton Dickinson, New Jersey, USA) were inoculated and incubated twice overnight at 35ºC. All morphologically suspected S. aureus isolates were identified using MALDI-TOF. A cefoxitin disk diffusion was performed on a Mueller Hinton agar (Becton Dickinson, New Jersey, USA). A growth inhibition zone of <22mm was considered resistant and confirmatory for MRSA. From the incubated vancomycin broth, a CHROMID® CARBA SMART Agar (bioMérieux, Marcy-l’Etoile, France), and an ESBL plate (Oxoid, Basingstoke, UK) were inoculated and incubated twice overnight at 35ºC. All morphologically different colonies were identified to species level using MALDI-TOF. For P. aeruginosa, A. baumannii, and Enterobacterales isolates growing on the CARB side of the CHROMID® CARBA SMART agar, a PCR was performed to detect blaVIM, blaIMP, blaNDM, blaKPC and blaOXA-48-like genes using established 152 Chapter 3.2
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