Environmental samples Environmental samples were taken 17 times at different intervals over a three year period, in both the old and the new hospital building (Figure 1) (11). In the old building, samples were taken twice, in the new building, samples were taken at 15 moments, from two weeks before to 36 months after relocation (Figure 1) (11). Different locations in patient rooms and bathrooms were sampled (Supplementary file 1) (11). Samples were taken with cotton swabs (BSN medical, Almere, the Netherlands), pre-moistened with PBS. Microbiological methods For nasal samples, 800µL swab medium was pipetted in a 6.5% NaCl TSB and incubated for 24 hours at 35°C. For environmental samples, swabs were placed in a 75mg/L aztreonam TSB directly after sampling, and incubated overnight at 35°C. After incubation, nasal and environmental samples were processed as follows: A PCR was performed to identify the S. aureus nucA- and mecA/mecC genes (Supplementary file 2). When S. aureus was identified, a blood agar was inoculated and incubated twice overnight at 35°C. The MALDI-TOF Biotyper (Matrix Assisted Laser Desorption Ionization-Time of Flight, Bruker Daltonics, Bremen, Germany) was used for the identification of all morphologically different suspected colonies. To determine beta-lactam antibiotic resistance, a cefoxitin disk diffusion test was performed. A growth inhibition zone of <22mm after 18-24 hours was considered resistant (12). Isolates were stored at -80°C. Clinical samples were processed according to routine diagnostic protocols as above. Staphylococcal protein A typing All included isolates were analyzed by spa-typing using established procedures (13). One nasal isolate per patient, one clinical isolate per patient, and one environmental isolate per location per spa type was included for the calculation of Simpsons’ index of diversity, which was used to assess the diversity within the population(s) (14). Definitions Colonization was defined as having a nasal screening sample with S. aureus at both admission and discharge, with both S. aureus being of the same spa type. Acquisition was defined as a negative culture at admission and a positive S. aureus culture at discharge, or when the discharge isolate was not identical to the admission strain. Loss of nasal carriage was defined as a positive culture at admission and a negative culture at discharge. When the clinical isolate was identical to the nasal isolate, the clinical isolate was considered endogenous. When the clinical isolate was not identical to the nasal isolate, the clinical isolate was considered acquired/exogenous. Statistical analyses Descriptive analyses were performed. For continuous variables, medians with range are presented. Normal distributed variables were analyzed with independent sample t-tests. 2 101 Dynamics of S. aureus in patients and the hospital environment
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