Presence of highly resistant microorganisms in the environment In the old building, 49 HRMO isolates were identified from 24 of the 724 (3.3%) sampled sites (Table 2). Thirty-seven out of 49 (75.5%) isolates were identified from patient rooms, not the ante-room or bathroom, and 44 out of 49 (89.8%) isolates were identified on the top or bottom of the sink plug (Table 2). In the new building, seven HRMO isolates were identified from five of the 4269 (0.1%) sampled sites, a significant decrease compared to the old building (P<0.001) (Table 2). All seven isolates were identified in the patient bathroom, five (71.4%) were identified from the shower drain (Table 2). In the new building, no HRMO were identified from the top or bottom of sink plugs (Table 2). In the old hospital building, 16 ESBL-E isolates were identified on 15 sample sites (eight Enterobacter spp., five Citrobacter spp., three Klebsiella spp.), 24 CP-PA isolates on 13 sample sites, and nine CPE isolates on five sample sites (four C. freundii isolates on three locations and five Enterobacter spp. isolates on three locations) (Table 2). In the new building, we identified three VRE isolates on three sample sites, three CPE isolates on one location (E. hormaechei) and one ESBL-E isolate on one sample site (K. pneumoniae) (Table 2). The three VRE positive locations were all identified in the same bathroom, one week after relocating. In both hospital buildings, no MRSA and CR-AB were detected. WGS was performed on all strains. Unfortunately, due to human error, we were unable to link the results of the WGS of isolates identified in the old hospital building to the locations where the isolates were found. Details of the analysis of the isolates were shown in Supplementary file 4. Most noteworthy, in CP-PA isolates a blaVIM-2 gene was detected, whereas in carbapenem-resistant C. freundii it involved a blaKPC-2 gene and in carbapenemresistant Enterobacter spp. a blaOXA-48 gene was detected. AmpC type beta-lactamase genes (e.g. blaCMY and blaDHA) were most often found in C. freundii (6 out of 8 isolates). In this relatively small collection of isolates, seven isolates (two C. freundii and five E. asburiae) contained a mcr-9 variant gene, but this involved several clonally related isolates. Upon clone correction this involved 3 strains. Three mcr-9 positive isolates had a minimum inhibitory concentration (MIC) of 0.5 µg/mL, and four strains had an MIC of 8 µg/mL, as measured by Vitek2. No other mcr genes were detected. In isolates that were considered to be genetically closely related, variation in the presence of AMR genes was detected. 3 159 Environmental contamination with MDRO in single-occupancy rooms
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